JP7339302B2 - Method for sequencing nucleic acids using time-resolved luminescence - Google Patents

Description

The present application relates generally to methods, compositions, and devices for performing rapid, massively parallel, quantitative analysis of biological and/or chemical samples, and methods for manufacturing such devices.

Detection and analysis of biological samples can be performed using biological assays (“bioassays”). Traditionally, bioassays involve large and expensive laboratory equipment, and such equipment requires researchers trained to operate the equipment and perform bioassays. Furthermore, bioassays are conventionally performed in bulk, requiring large amounts of specific types of samples for detection and quantification.

Some bioassays are performed by tagging samples with luminescent markers that emit light at specific wavelengths. A marker is illuminated with a light source to cause it to emit light, and the emitted light is detected by a photodetector to quantify the amount of emitted light emitted by the marker. Bioassays using luminescent markers traditionally involve an expensive laser light source to illuminate the sample and complex emission detection optics and electronics to collect the emission from the illuminated sample.

FIG. 1 depicts a non-limiting schematic representation of a sample well (eg, sample well) containing various components for nucleic acid sequencing. Diagram representing a non-limiting exemplary experiment of four-step nucleic acid sequencing; (A) before incorporation of luminescence-labeled nucleotide; (B) first integration event; (C) first and second integration events. and (D) a second integration event; and examples of corresponding raw and processed data during stages (A) through (D). FIG. 3 shows the normalized cumulative distribution functions for unrestricted signal versus emission time and probability of decay for four luminescent molecules with different emission lifetimes. FIG. 3 depicts non-limiting luminescence lifetime charts and luminescence intensity charts for exemplary luminescence-labeled nucleotides. Diagram showing a non-limiting sequencing experiment with four luminescence-labeled nucleotides: (A) Traces of detected luminescence from green and red pulses; (B) Green based on luminescence lifetime and intensity of each nucleotide incorporation. data reduction from the pulse; and (C) alignment of the experimentally determined sequence with the template sequence. Figures showing a non-limiting sequencing experiment using four luminescently labeled nucleotides: (A) Traces of detected luminescence from green pulses; (B) from green pulses based on luminescence lifetime and intensity of each nucleotide incorporation. and (C) alignment of the experimentally determined sequence with the template sequence. Diagram representing a non-limiting exemplary surface preparation process including the steps of (a) deposition, (b) passivation, (c) silicification, (d) complex loading, and (e) sequencing reaction initiation. Schematic representation of two non-limiting methods for passivating metal oxide surfaces with phosphonate-PEG groups. Diagram representing two non-limiting methods for functionalization of glass with silane-PEG-biotin and silane-PEG mixtures. FIG. 3 shows a non-limiting example of one or more luminescent molecules and one or more nucleotides linked via a protecting molecule. FIG. 11 shows non-limiting examples of configurations comprising one or more nucleotides and one or more luminescent molecules attached to a protective molecule via a split linker. Non-limiting Embodiments of Configurations of Luminescently Labeled Nucleotide Molecules that May be Used in Sequencing Reactions and Exemplary Sequencing Experiments in which the Luminescent Properties of Luminescent Labeled Nucleotides are Used to Identify Bases Incorporated into Sequencing Reactions A diagram showing the Non-limiting Embodiments of Configurations of Luminescently Labeled Nucleotide Molecules that May be Used in Sequencing Reactions and Exemplary Sequencing Experiments in which the Luminescent Properties of Luminescent Labeled Nucleotides are Used to Identify Bases Incorporated into Sequencing Reactions A diagram showing the Non-limiting Embodiments of Configurations of Luminescently Labeled Nucleotide Molecules that May be Used in Sequencing Reactions and Exemplary Sequencing Experiments in which the Luminescent Properties of Luminescent Labeled Nucleotides are Used to Identify Bases Incorporated into Sequencing Reactions A diagram showing the Non-limiting examples of designing non-functional binding sites to protective molecules for binding light-emitting molecules and nucleotides at selected sites. Non-limiting examples of designing non-functional binding sites to protective molecules for binding light-emitting molecules and nucleotides at selected sites. Non-limiting examples of designing non-functional binding sites to protective molecules for binding light-emitting molecules and nucleotides at selected sites. FIG. 4 shows a non-limiting example in which a luminescent molecule and a nucleotide are connected via a protecting molecule, where the linker connecting the luminescent molecule and nucleotide to the protecting molecule comprises an oligonucleotide. Figure 4 shows a non-limiting example in which a luminescent molecule is attached to a protective molecule via annealed complementary oligonucleotides, where each oligonucleotide strand has one non-specific binding site that matches a binding site on the protective molecule. Diagram with covalent ligands. Figure 4 shows a non-limiting example in which a luminescent molecule is attached to a protective molecule via annealed complementary oligonucleotides, where each oligonucleotide strand has one non-specific binding site that matches a binding site on the protective molecule. Diagram with covalent ligands. Non-limiting examples of utilizing genetically encoded tags to attach luminescent molecules and nucleotides to discrete sites on protective molecules. Non-limiting examples of utilizing genetically encoded tags to attach luminescent molecules and nucleotides to discrete sites on protective molecules. Figure 2 shows a non-limiting example of utilizing reactive groups at protein end groups to attach luminescent molecules and nucleotides to independent sites on protective molecules. Figure 2 shows a non-limiting example of utilizing reactive groups at protein end groups to attach luminescent molecules and nucleotides to independent sites on protective molecules. Figure 2 shows a non-limiting example of utilizing reactive groups at protein end groups to attach luminescent molecules and nucleotides to independent sites on protective molecules. Figure 2 shows a non-limiting example of utilizing unnatural amino acids to attach light-emitting molecules and nucleotides to independent sites on protective molecules. Figure 2 shows a non-limiting example of utilizing unnatural amino acids to attach light-emitting molecules and nucleotides to independent sites on protective molecules. Figure 2 shows a non-limiting example of utilizing unnatural amino acids to attach light-emitting molecules and nucleotides to independent sites on protective molecules. Figure 2 shows a non-limiting example of utilizing unnatural amino acids to attach light-emitting molecules and nucleotides to independent sites on protective molecules. Figure 2 shows a non-limiting example of utilizing unnatural amino acids to attach light-emitting molecules and nucleotides to independent sites on protective molecules. A non-limiting example of a luminescent molecule and a nucleotide separated by a non-protein protecting molecule. A non-limiting example of a luminescent molecule and a nucleotide separated by a non-protein protecting molecule. FIG. 1 shows a non-limiting embodiment of a protective molecule consisting of a protein-protein binding pair. FIG. 11 shows non-limiting examples of linker configurations. Non-limiting reaction schemes and exemplary structures for nucleotide linker synthesis. Non-limiting reaction schemes and exemplary structures for nucleotide linker synthesis. FIG. 2 is a block diagram representation of an apparatus that can be used for rapid mobility analysis of biological and chemical samples, according to some embodiments; 1 is a block diagram of an integrated device and equipment, according to some embodiments; FIG. FIG. 4 illustrates a row of pixels of an integrated device, according to some embodiments; FIG. 4 illustrates coupling of excitation energy to sample wells in a row of pixels and radiant energy from each sample well directed at the sensor, according to some embodiments. FIG. 3 illustrates an integrated device and excitation source according to some embodiments; FIG. 3 illustrates an integrated device and excitation source according to some embodiments; FIG. 4 illustrates a sample well formed within a pixel region of an integrated device according to one embodiment. FIG. 4 illustrates excitation energy incident on a sample well, according to some embodiments. FIG. 10 illustrates a sample well that, in some embodiments, includes a divot that increases excitation energy in an excitation region associated with the sample well. FIG. 10 illustrates sample wells and divots according to some embodiments; FIG. 4 illustrates a sensor with time bins, according to some embodiments; FIG. 4 illustrates a sensor with time bins, according to some embodiments; 1 illustrates an exemplary system for providing light pulses according to some embodiments; FIG. FIG. 4 shows a plot of light intensity as a function of time; FIG. 4 shows a schematic diagram for taking measurements, according to some embodiments. FIG. 4 shows a plot of light signal as a function of time, according to some embodiments; FIG. 10 shows signal profiles of markers across time bins, according to some embodiments. FIG. 4 shows a plot of light signal as a function of time, according to some embodiments; FIG. 10 shows signal profiles of markers across time bins, according to some embodiments. FIG. 4 shows a schematic diagram for taking measurements, according to some embodiments. FIG. 4 shows a plot of lifetime as a function of emission wavelength, according to some embodiments; FIG. 4 shows a plot of optical signal as a function of wavelength, according to some embodiments; FIG. 4 shows a plot of light signal as a function of time, according to some embodiments; FIG. 11 shows signal profiles of markers across time bins for multiple sensors, according to some embodiments. FIG. 4 shows a plot of light signal as a function of time, according to some embodiments; FIG. 11 shows signal profiles of markers across time bins for multiple sensors, according to some embodiments. FIG. 4 shows a schematic diagram for performing measurements, according to some embodiments; FIG. 4 shows a plot of optical signal as a function of wavelength, according to some embodiments; FIG. 4 shows a plot of light signal as a function of time, according to some embodiments; FIG. 10 shows signal profiles of markers across multiple sensor time bins, according to some embodiments.

According to one aspect of the present application, a single molecule can be identified based on one or more properties of a sequence of photons emitted from said molecule when exposed to a plurality of separate light pulses. It is possible (eg, it can be distinguished from other possible molecules in the reaction sample). In some embodiments, emitted photons can be used to identify/discriminate molecules based on their different emission lifetimes. In some embodiments, a luminescence lifetime can be calculated. In some embodiments, the actual lifetime may not be calculated. For example, one or more properties indicative of luminescence lifetime may be used (eg, local time of radiation, time distribution of detected radiation). In some embodiments, emitted photons may be used to determine the emission intensity of the molecule, and the emission intensity may be used to identify the molecule. In some embodiments, emitted photons may be used to determine the luminescence lifetime and luminescence intensity of a molecule, and the luminescence lifetime or luminescence intensity may be used to identify the molecule. In some embodiments, molecular identity may be based on a combination of both emission lifetime and emission intensity.

In some embodiments, the molecule may be labeled with a luminescent label. In some embodiments, the luminescent label is a fluorophore. In some embodiments, luminescent labels can be identified or distinguished based on properties of the luminescent label. Properties of luminescent labels (eg, fluorophores) include, but are not limited to, emission lifetime, absorption spectrum, emission spectrum, emission quantum yield, and emission intensity. In some embodiments, luminescent labels are identified or distinguished based on their luminescent lifetime. In some embodiments, luminescent labels are identified or distinguished based on luminescence intensity. In some embodiments, luminescent labels are identified or distinguished based on the wavelength of applied excitation energy required to observe emitted photons. In some embodiments, luminescent labels are identified or distinguished based on the wavelength of emitted photons. In some embodiments, luminescent labels are identified or distinguished based on both the luminescent lifetime and the wavelength of applied excitation energy required to observe emitted photons. In some embodiments, luminescent labels are identified or distinguished based on both the emission intensity and the wavelength of applied excitation energy required to observe emitted photons. In some embodiments, luminescent labels are identified or differentiated based on the emission lifetime, emission intensity, and wavelength of applied excitation energy required to observe emitted photons. In some embodiments, luminescent labels are identified or distinguished based on both luminescent lifetime and wavelength of emitted photons. In some embodiments, luminescent labels are identified or distinguished based on both the intensity of the emitted light and the wavelength of the emitted photons. In some embodiments, luminescent labels are identified or distinguished based on luminescent lifetime, luminescent intensity, and wavelength of emitted photons.

In certain embodiments, different types of molecules in a reaction mixture or experiment are labeled with different luminescent markers. In some embodiments, different markers have different luminescence properties that can be discriminated. In some embodiments, different markers are distinguished by having different emission lifetimes, different emission intensities, different wavelengths of emitted photons, or combinations thereof. The presence of multiple types of molecules with different luminescent markers can allow different steps of a conjugation reaction to be monitored, or different components of conjugation reaction products to be identified. In some embodiments, the order in which different types of molecules react or interact can be determined.

In some embodiments, different nucleotides may be luminescently labeled with different numbers of the same luminescent molecule (eg, with one or more of the same fluorochromes). In some embodiments, varying the number of luminescent molecules of a luminescent label can allow different nucleotides to be distinguished based on different intensities. In some embodiments, each different nucleotide may be labeled with a different type of luminescent molecule and/or a different number of each luminescent molecule. In some embodiments, different types of luminescent molecules allow different nucleotides to be distinguished based on different intensities and/or different lifetimes.

In some embodiments, the luminescent properties of multiple types of molecules with different luminescent markers are used to distinguish sequences of biomolecules such as nucleic acids or proteins. In some embodiments, the luminescent properties of multiple types of molecules with different luminescent markers are used to identify single molecules as they are incorporated during the synthesis of biomolecules. In some embodiments, the luminescent properties of multiple types of nucleotides with different luminescent markers are used to distinguish single nucleotides when they are incorporated into a sequencing reaction. This may allow determination of the sequence of the nucleic acid template. In some embodiments, the luminescently labeled nucleotide is incorporated into a nucleic acid strand complementary to the nucleic acid template. In some embodiments the complementary strand comprises a primer.

In certain embodiments, the multiple types of nucleotides with different luminescent markers comprise four types of luminescently labeled nucleotides. In some embodiments, the four luminescently labeled nucleotides absorb and/or emit photons within one spectral range. In some embodiments, three of the luminescently labeled nucleotides emit in a first spectral range and a fourth luminescently labeled nucleotide absorbs and/or emits photons in a second spectral range. In some embodiments, two of the luminescently labeled nucleotides emit in a first spectral range and the third and fourth luminescently labeled nucleotides emit in a second spectral range. In some embodiments, two of the luminescently labeled nucleotides emit within a first spectral range, a third nucleotide absorbs and/or emits photons within a second spectral range, and a fourth Nucleotides absorb and/or emit photons within the third spectral range. In some embodiments, each of the four luminescently labeled nucleotides absorbs and/or emits photons within different spectral ranges. In some embodiments, each type of luminescently labeled nucleotide that absorbs and/or emits photons within the same spectral range has a different emission lifetime or emission intensity or both.

In some embodiments, each of the four different types of nucleotides (eg, adenine, guanine, cytosine, thymine/uracil) in the reaction mixture may be labeled with one or more luminescent molecules. In some embodiments, each type of nucleotide may be attached to more than one of the same luminescent molecule (eg, two or more of the same fluorescent dyes are attached to the nucleotide). In some embodiments, each luminescent molecule may be attached to more than one nucleotide (eg, two or more of the same nucleotide). In some embodiments, more than one nucleotide may be attached to more than one luminescent molecule. In some embodiments, the protective molecule serves as an anchor for attaching one or more nucleotides (e.g., of the same type) and one or more luminescent molecules (e.g., of the same type). . In some embodiments, all four nucleotides are labeled with luminescent molecules that absorb and emit within the same spectral range (eg, 520-570 nm).

In some embodiments, the luminescent labels in a set of four nucleotides can be selected from dyes comprising aromatic or heteroaromatic compounds, pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, carbazole, thiazole, benzothiazole, phenanthridine, phenoxazine, porphyrin, quinoline, ethidium, benzamide, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluoroscein, rhodamine, or other similar compounds. Exemplary dyes include xanthene dyes such as fluorescein dyes or rhodamine dyes, naphthalene dyes, coumarin dyes, acridine dyes, cyanine dyes, benzoxazole dyes, stilbene dyes, pyrene dyes, phthalocyanine dyes, phycobiliprotein dyes, and squaraines. A dye etc. are mentioned.

In some embodiments, the luminescent label in the set of four nucleotides is Alexa Fluor® 546, Cy® 3B, Alexa
Fluor® 555, and Alexa Fluor® 555, and the FRET pair Alexa Fluor® 555 and Cy® 3.5. In some embodiments, the luminescent label in the set of four nucleotides is Alexa Fluor® 555, Cy® 3.5, Alexa
Fluor® 546, and DyLight® 554-R1. In some embodiments, the luminescent labels in the set of four nucleotides are Alexa Fluor® 555, Cy® 3.5, ATTO Rho 6G, and DyLight® 554 - includes R1. In some embodiments, the luminescent label in the set of four nucleotides comprises Alexa Fluor® 555, Cy® 3B, ATTO Rho6G, and DyLight® 554-R1. In some embodiments, the luminescent label in the set of four nucleotides comprises Alexa Fluor® 555, Cy® 3B, ATTO542, and DyLight® 554-R1. In some embodiments, the luminescent labels in the set of four nucleotides include Alexa Fluor® 555, Cy® 3B, ATTO542, and Alexa Fluor® 546. In some embodiments, the luminescent label in the set of four nucleotides comprises Cy®3.5, Cy®3B, ATTO Rho6G, and DyLight®554-R1.

In certain embodiments, at least one type, at least two types, at least three types, or at least four of the types of luminescently labeled nucleotides are 6-TAMRA, 5/6-Carboxyrhodamine 6G, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 610, Alexa Fluor® 647, Aberrior Star 635, ATTO ) 425, ATTO465, ATTO488, ATTO495, ATTO514, ATTO520, ATTO Rho 6G, ATTO542, ATTO647N, ATTO Rho14, Chromis 630, Chromis 654A, Chromeo™ 642, CF (trademark) 514, CF(TM) 532, CF(TM) 543, CF(TM) 546, CF(TM) 546, CF(TM) 555, CF(TM) 568, CF(TM) 633, CF(TM) 640R, CF( Trademark) 660C, CF(TM) 660R, CF(TM) 680R, Cy(R) 3, Cy(R) 3B, Cy(R) 3.5, Cy(R) 5, Cy(R) ) 5.5, Dyomics-530, Dyomics-547P1, Dyomics-549P1, Dyomics-550, Dyomics-554, Dyomics-555, Dyomics-556, Dyomics-560, Dyomics-650, Dyomics- 680, DyLight ( 554-R1, DyLight ® 530-R2, DyLight ® 594, DyLight ® 635-B2, DyLight ® 650, DyLight ® 655-B4, DyLight ( 675-B2, DyLight® 675-B4, DyLight® 680, HiLyte™ Fluor 532, HiLyte™ Fluor 555, HiLyte™ Fluor 594, Light selected from the group consisting of LightCycler® 640R, Seta™ 555, Seta™ 670, Seta™ 700, Seta™ u647, and Seta™ u665 It comprises a luminescent dye or is of the formulas (Dye 101), (Dye 102), (Dye 103), (Dye 104), (Dye 105), or (Dye 106) described herein.

In some embodiments, at least one type, at least two types, at least three types, or at least four of the types of luminescently labeled nucleotides are each Alexa Fluor® 546, Alexa Fluor® 555, Cy® 3B, Cy 3.5, DyLight® 554-R1, Alexa Fluor® 546, ATTO Rho6G, ATTO425, ATTO465, ATTO488, ATTO495, ATTO514, It comprises a luminescent dye selected from the group consisting of ATTO520, ATTO Rho6G, and ATTO542.

In some embodiments, the first type of luminescent labeled nucleotide comprises Alexa Fluor® 546, the second type of luminescent labeled nucleotide comprises Cy® 3B, and the third type includes two Alexa Fluor® 555, and a fourth type of luminescent labeled nucleotide includes Alexa Fluor® 555 and Cy® 3.5.

In some embodiments, the first type of luminescence-labeled nucleotide comprises Alexa Fluor® 555, the second type of luminescence-labeled nucleotide comprises Cy® 3.5, the third A type of luminescence-labeled nucleotide includes Alexa Fluor® 546 and a fourth type of luminescence-labeled nucleotide includes DyLight® 554-R1.

In some embodiments, the first type of luminescence-labeled nucleotide comprises Alexa Fluor® 555, the second type of luminescence-labeled nucleotide comprises Cy® 3.5, the third A type of luminescence-labeled nucleotide includes ATTO Rho6G and a fourth type of luminescence-labeled nucleotide includes DyLight® 554-R1.

In some embodiments, the first type of luminescent-labeled nucleotide comprises Alexa Fluor® 555, the second type of luminescent-labeled nucleotide comprises Cy® 3B, and the third type of includes ATTO Rho6G and a fourth type of luminescent labeled nucleotide includes DyLight® 554-R1.

In some embodiments, the first type of luminescent-labeled nucleotide comprises Alexa Fluor® 555, the second type of luminescent-labeled nucleotide comprises Cy® 3B, and the third type of includes ATTO542, and a fourth type of luminescent labeled nucleotide includes DyLight®554-R1.

In some embodiments, the first type of luminescent-labeled nucleotide comprises Alexa Fluor® 555, the second type of luminescent-labeled nucleotide comprises Cy® 3B, and the third type of includes ATTO542 and a fourth type of luminescent labeled nucleotide includes Alexa Fluor®546.

In some embodiments, the first type of luminescent-labeled nucleotide comprises Cy®3.5, the second type of luminescent-labeled nucleotide comprises Cy®3B, and the third type of Types of luminescence-labeled nucleotides include ATTO Rho6G and a fourth type of luminescence-labeled nucleotides includes DyLight® 554-R1.

In some embodiments, at least one type, at least two types, at least three types, or at least four of the types of luminescently labeled nucleotides are Alexa Fluor® 532, Alexa Fluor® ) 546, Alexa Fluor® 555, Alexa Fluor® 594, Alexa Fluor® 610, CF™ 532, CF™ 543, CF™ 555, CF™ 594 , Cy® 3, DyLight® 530-R2, DyLight® 554-R1, DyLight® 590-R2, DyLight® 594, DyLight® 610-B1 or of the formula (dye 101), (dye 102), (dye 103), (dye 104), (dye 105), or (dye 106).

In some embodiments, the first and second types of luminescently labeled nucleotides are Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, CF™ 532, A third and a fourth type of luminescent labeled nucleotides are Alexa Fluor® 594, Alexa Fluor® 610, CF™ 594, DyLight® 590-R2, DyLight® 594, DyLight (Dye 101), (Dye 102), (Dye 103), (Dye 104), (Dye 105), or (Dye 106).

In certain embodiments, at least one type, at least two types, at least three types, or at least four of the types of luminescently labeled nucleotide molecules are TagBFP, mTagBFP2, Azurite, EBFP2, mKalama1, Sirius (SIRIUS), sapphire, T -SAPPHIRE, ECFP, Cerulean (Cerulean), SCFP3A, MTURQUOOISE, MTURQUOOISE2, single quantity MIDORIISHI -CYAN, TAGCFP, MTFP1, EGFP, Emerald, Super Folder (SUPERFOLDER) GFP , monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, mNeonGreen, EYFP, Citrine, Venus, SYFP2, TagYFP, monomeric Kusabira-Orange ), mKOK, mKO2, mOrange, mOrange2, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mRuby2, mPlum, HcRed-Tandem, mK ate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKate1, LSS-mKate2, mBeRFP, PA-GFP, PAmCherry1, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS - containing a photoprotein selected from the group consisting of CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

Aspects of the present application provide methods for applying excitation energy to molecules to be identified and detecting emitted photons after excitation, as well as systems and devices useful in such methods. In certain embodiments, detecting includes recording, for each detected luminescence, the elapsed time between the luminescence and the prior pulse of excitation energy. In certain embodiments, detecting includes recording, for each of the plurality of detected emissions, the elapsed time between the emission and the prior pulse of excitation energy. In some embodiments, multiple pulses of excitation energy are applied. A luminescent marker of the molecule to be identified can be excited by each of the pulses or portions thereof. In some embodiments, multiple emissions are detected by one or more sensors. A luminescent marker of a molecule to be identified may emit luminescence after each excitation or portion of the excitation. The rate of excitation events that result in luminescence is based on the luminescence quantum yield of the marker. In some embodiments, increasing the number of luminescent molecules comprising a luminescent marker can increase the quantum yield (eg, can increase the number of luminescent emissions). Additionally, not all luminescence emitted by the marker will be detected. For example, some emissions will be directed away from the sensor. In certain embodiments, one or more excitation energies are selected based on emission properties of the luminescent marker, including absorption spectra and wavelengths at which the marker emits photons upon excitation in a given spectral range.

In certain embodiments, the frequency of pulsed excitation energy is selected based on the emission properties (eg, emission lifetime) of the luminescent label molecule to be detected. In some embodiments, the interval between pulses is longer than the emission lifetime of the one or more luminescent labeled molecules being excited. In some embodiments, the spacing is about 2 to about 10 times, about 10 to about 100 times, or about 100 to about 10 times longer than the emission lifetime of one or more luminescent label molecules being excited. 1000 times longer. In some embodiments, the interval is about 10 times longer than the emission lifetime of the one or more luminescent labeled molecules being excited.

In some embodiments, the frequency of pulsed excitation energy is selected based on the chemical process being monitored. For sequencing reactions, the number of pulses applied to the target volume during incorporation of luminescently labeled nucleotides will partially determine the number of emitted photons detected. In some embodiments, the frequency is selected such that a sufficient number of photons can be detected during incorporation of the luminescently labeled nucleotides, a sufficient number being among multiple types of luminescently labeled nucleotides. , is the number of photons required to discriminate that luminescently labeled nucleotide. In some embodiments, luminescently labeled nucleotides are distinguished based on the wavelength of emitted photons. In some embodiments, luminescent-labeled nucleotides are distinguished based on their luminescent radiative lifetime, eg, the time between pulsed excitation and radiative detection. In some embodiments, a luminescently labeled nucleotide is distinguished based on the wavelength of the emitted photon and the emission radiative lifetime. In some embodiments, a luminescently labeled nucleotide is distinguished based on the luminescent intensity of the emitted signal (eg, based on the frequency of emission or the total number of emitted events within a period of time). In some embodiments, luminescently labeled nucleotides are distinguished based on the emission intensity and emission lifetime of the emitted signal. In some embodiments, luminescently labeled nucleotides are distinguished based on emission intensity and wavelength. In some embodiments, luminescent-labeled nucleotides are distinguished based on luminescence intensity, wavelength, and luminescence lifetime.

According to one aspect of the present application, a system is provided that includes an excitation source module and an integrated device. The excitation source module includes an excitation source configured to emit pulses of excitation energy having a first duration. The integrated device is configured to receive a target volume that emits luminescence when coupled with a pulse of excitation energy; the pulse of excitation energy travels from the energy source coupling component to the target volume. a sensor detecting luminescence over a second duration longer than said first duration; a second energy pathway in which luminescence travels from the target volume to the sensor; and a pulse of excitation energy is , including a third energy path traveling from the excitation source to the energy source coupling component.

According to another aspect of the present application, an integrated device comprising a target volume and a sensor is provided. The target volume is configured to receive a sample labeled with one of a plurality of luminescent markers each having a different lifetime value. A sensor is configured to detect luminescence from one of the plurality of luminescent markers over a plurality of elapsed times. Multiple elapsed times are selected to distinguish multiple luminescent markers.

According to another aspect of the present application, an integrated device comprising a target volume and multiple sensors is provided. A target volume is configured to receive a sample labeled with one of a plurality of luminescent markers. Each of the plurality of luminescent markers emits luminescence within one of the plurality of spectral ranges, and portions of the plurality of luminescent markers that emit luminescence in one of the plurality of spectral ranges each have a different luminescence lifetime. . Each sensor of the plurality of sensors is configured to detect one of the plurality of spectral ranges over a plurality of elapsed times, the plurality of elapsed times selected to distinguish portions of the plurality of luminescent markers. .

According to another aspect of the present application, a system is provided that includes multiple excitation sources and an integrated device. Multiple excitation sources emit multiple excitation energies. Each of the multiple excitation sources emits one pulse of multiple excitation energies. The integrated device comprises a target volume configured to receive a sample labeled with one of a plurality of luminescent markers; A sensor configured to detect luminescence from one of the luminescent markers is included. Each portion of the plurality of luminescent markers that emits luminescence after being illuminated by one of the plurality of excitation energies has a different lifetime value. Accumulation of multiple data timings for the duration between the pulse event and the luminescence emission distinguishes multiple luminescence markers.

According to another aspect of the application, a method is provided for sequencing a target nucleic acid. In some embodiments, a method for sequencing a target nucleic acid comprises the steps of: (i) (a) said target nucleic acid, (b) primers complementary to said target nucleic acid, (c) nucleic acid providing a mixture comprising a polymerase and (d) nucleotides for incorporation into a growing nucleic acid strand complementary to said target nucleic acid, said nucleotides comprising different types of luminescent labeled nucleotides, said luminescent labeled Nucleotides produce a detectable signal during sequential incorporation into the growing nucleic acid strand, and detectable signals from the different types of luminescently labeled nucleotides are distinguishable from each other in the time domain (e.g., detection by determining the timing and/or frequency of possible signals), step (ii) under conditions sufficient to produce said growing nucleic acid strand by extending said primer, said subjecting the mixture to a polymerization reaction, (iii) measuring the detectable signal from the luminescently labeled nucleotides during sequential incorporation into the growing nucleic acid strand, and (iv) the growing nucleic acid strand. determining the timing and/or frequency of the measured detectable signal from the luminescent-labeled nucleotides upon sequential incorporation into the growing nucleic acid strand to identify the timeline of incorporation of the luminescent-labeled nucleotides into the growing nucleic acid strand; , thereby determining the sequence of said target nucleic acid.

In some embodiments, the target nucleic acid or nucleic acid polymerase is attached to a support. In some embodiments, the time series of incorporation is identified after subjecting the mixture of (i) to a polymerization reaction. In some embodiments the detectable signal is an optical signal. In some embodiments, the optical signal is a luminescent signal. In some embodiments, determining the timing and/or frequency of the measured detectable signal comprises the steps of: (i) receiving said detectable signal at one or more sensors; and (ii) said one charge carriers of a plurality of charge carriers generated in response to the detectable signals received at one or more sensors into at least one charge carrier storage area based on the time the charge carriers were generated; and the step of selectively directing.

In some embodiments, the measured timing and/or frequency of the detectable signal comprises a decay lifetime measurement. In some embodiments, the measured timing and/or frequency of the detectable signal comprises a measurement of the time of arrival of the detectable signal at one or more sensors that detect the detectable signal. In some embodiments, the method comprises separating charge carriers generated by the detectable signal into bins associated with one or more sensors based on time of arrival of the detectable signal. further provide. In some embodiments, the measured timing and/or frequency of detectable signals are non-spectral measurements.

Those skilled in the art will understand that the drawings described herein are for illustration only. In some cases, it should be understood that various aspects of the invention may be exaggerated or exaggerated in order to facilitate understanding of the invention. In the drawings, like reference numbers generally refer to like features, functionally similar and/or structurally similar elements throughout the various views. The drawings are not necessarily to scale, emphasis rather being placed upon illustrating the principles of teaching. The drawings are in no way intended to limit the scope of the present teachings.

The features and advantages of the present invention will become more apparent from the detailed description set forth below when taken in conjunction with the drawings.
When describing embodiments with reference to the drawings, directional references (such as “top,” “bottom,” “top,” “bottom,” “left,” “right,” “horizontal,” “vertical,” etc.) are used. may be used. Such references are merely intended to assist the reader in viewing the drawings in their normal orientation. These directional references are not intended to describe preferred or sole orientations of embodied devices. The device may be embodied in other orientations.

As will become apparent from the detailed description, the examples shown in the drawings (e.g., FIGS. 1-37) and further described for purposes of illustration throughout this application describe non-limiting embodiments, In some cases, certain processes may be simplified or features or steps omitted for the purpose of more clear illustration.

(detailed explanation)
The inventors have developed new methods, compositions, and devices for identifying single molecules based on one or more of their luminescent properties. In some embodiments, molecules are identified based on their emission lifetime, absorption spectrum, emission spectrum, emission quantum yield, emission intensity, or a combination of two or more thereof. Identification may mean ascribed the exact molecular identity of a molecule, or it may mean discriminating or distinguishing a particular molecule from a set of possible molecules. In some embodiments, molecules can be distinguished from each other based on different emission lifetimes, absorption spectra, emission spectra, emission quantum yields, emission intensities, or combinations of two or more thereof. In some embodiments, single molecules are identified by exposing the molecule to a series of discrete light pulses and assessing the timing or other properties of each photon emitted from the molecule (e.g., other molecules). In some embodiments, the information of multiple photons emitted sequentially from a single molecule is integrated and evaluated to identify the molecule. In some embodiments, the luminescence lifetime of a molecule is determined from multiple photons sequentially emitted from the molecule, and the luminescence lifetime can be used to identify the molecule. In some embodiments, the emission intensity of a molecule is determined from multiple photons sequentially emitted from the molecule, and the emission intensity can be used to identify the molecule. In some embodiments, the luminescence lifetime and luminescence intensity of a molecule are determined from a plurality of photons sequentially emitted from the molecule, and the luminescence lifetime and luminescence intensity can be used to identify the molecule. .

One aspect of the present application is useful for detecting and/or identifying one or more biological or chemical molecules. In some embodiments, a chemical or biological reaction can be assessed by determining the presence or absence of one or more reagents or products at one or more time points. be.

In one aspect of the present application, molecules are probed by exposing them to light and determining one or more properties of one or more photons emitted from the molecule. In certain aspects of the application, molecules are probed by exposing them to pulses of light and determining one or more properties of the photons emitted from the molecule. In some embodiments, the molecule is exposed to multiple individual light pulse events and one or more properties of the individual photons emitted after the individual light pulse events are determined. In some embodiments, the molecule does not emit photons in response to each light pulse. However, multiple emitted photons expose the molecule to a series of individual light pulses, with individual photons emitted after a subset of the light pulse events (e.g., photons emitted after about 10% of the pulse events). , or photons emitted after about 1% of the pulse event).

One aspect of the present application monitors chemical or biological reactions by determining the presence or absence of one or more reagents, intermediates, and/or products at one or more time points. It is useful to In some embodiments, the progress of the reaction over time is analyzed by exposing the reaction sample to a series of discrete light pulses and analyzing any emitted photons detected after each light pulse. is possible.

Thus, in some aspects of the present application, a reaction sample is exposed to multiple discrete pulses of light and a series of emitted photons are detected and analyzed. In some embodiments, the train of emitted photons provides information about single molecules present in the reaction sample that do not change over the time of the experiment. However, in some embodiments, a series of emitted photons provides information about a series of different molecules present in the reaction sample at different times (eg, as the reaction or process progresses).

In some embodiments, one aspect of the present application is to analyze a biological sample, e.g., to determine the sequence of one or more nucleic acids or polypeptides in the sample, and/or It can be used to determine the presence or absence of one or more nucleic acid or polypeptide variants (eg, one or more mutations of a gene of interest) in the medium. In some embodiments, patient samples (e.g., , human patient samples). In some instances, diagnostic tests include, for example, sequencing cell-free deoxyribonucleic acid (DNA) molecules and/or expression products (e.g., ribonucleic acid (RNA)) in a subject's biological sample. Sequencing nucleic acid molecules in the subject's biological sample.

In some embodiments, one or more molecules being analyzed (e.g., interrogated and/or identified) using luminescence lifetime and/or intensity are labeled molecules (e.g., one or more luminescence molecules labeled with markers). In some embodiments, individual subunits of biomolecules may be identified using markers. In some examples, luminescent markers may be used to identify individual subunits of biomolecules. In some embodiments, luminescent markers (also referred to herein as "markers") are used. A luminescent marker can be an exogenous marker or an endogenous marker. Exogenous markers may be exogenous luminescent markers used as reporters and/or tags for luminescent labels. Examples of exogenous markers include, but are not limited to, fluorescent molecules, fluorophores, fluorescent dyes, fluorescent dyes, organic dyes, fluorescent proteins, species that contribute to fluorescence resonance energy transfer (FRET), enzymes, and/or Quantum dots can be mentioned. Other exogenous markers are known in the art. Such exogenous markers may be conjugated to probes or functional groups (eg, molecules, ions, and/or ligands) that specifically bind to particular targets or moieties. Attaching an exogenous tag or reporter to the probe allows target identification by detection of the presence of the exogenous tag or reporter. Examples of probes can include protein, nucleic acid (eg, DNA, RNA) molecules, lipids, and antibody probes. Combinations of exogenous markers and functional groups include molecular probes, labeled probes, hybridization probes, antibody probes, protein probes (e.g., biotin-conjugated probes), enzymatic labels, fluorescent probes, fluorescent tags, and/or enzymatic reporters. Any suitable probes, tags, probes and/or labels used for detection can be formed.

Although this disclosure refers to luminescent markers, other types of markers may be used with the devices, systems, and methods provided herein. Such markers may be mass tags, electrostatic tags, electrochemical labels, or any combination thereof.

Exogenous markers may be added to the sample and endogenous markers may already be part of the sample. Endogenous markers can include any luminescent marker present that is capable of luminescence or "autofluorescence" in the presence of excitation energy. Autofluorescence of endogenous fluorophores can provide label-free, non-invasive labeling without the need for introduction of exogenous fluorophores. Examples of such endogenous fluorophores include hemoglobin, oxyhemoglobin, lipids, collagen and elastin crosslinks, reduced nicotinamide adenine dinucleotide (NADH), oxidized flavins (FAD and FMN), lipofuscin, keratin, and/or or porphyrins, which are given by way of example and not limitation.

The inventors recognize that there is a need for simpler and easier devices for performing single-molecule detection and/or nucleic acid sequencing. We have come up with techniques for single molecule detection that use markers, luminescent labels) to label different molecules. Such a single molecule may be a nucleotide or amino acid with a tag. A tag may be detected bound to a single molecule, detected upon release from that single molecule, or bound to and from a single molecule. may be detected as it is emitted from the In some examples, the tag is a luminescent tag. Each luminescent tag in the selected set is associated with a respective molecule. For example, a set of four tags may be used to "label" a nucleobase present in DNA. Each tag in the set is associated with a different nucleobase, e.g., the first tag is associated with adenine (A), the second tag is associated with cytosine (C), the second tag is associated with A third tag is associated with guanine (G) and a fourth tag is associated with thymine (T). Additionally, each of the light emitting tags in the set of tags has different properties that can be used to distinguish the first tag in the set from the other tags in the set. In this manner, each tag is uniquely identifiable using one or more of these distinguishing features. By way of example, and not limitation, tag characteristics that can be used to distinguish one tag from another include the characteristics emitted by the tag in response to excitation energy. the radiant energy and/or wavelength of the light that is emitted, the wavelength of the excitation light that is absorbed by a particular tag and places that tag in an excited state, and/or the radiative lifetime of that tag.

Sequencing Some aspects of the present application are useful for sequencing biological polymers such as nucleic acids and proteins. In some embodiments, the methods, compositions, and devices described in this application may be used to identify a series of nucleotide or amino acid monomers that are incorporated into a nucleic acid or protein (e.g., a series of by detecting the time course of incorporation of labeled nucleotide monomers or labeled amino acid monomers). In some embodiments, the methods, compositions, and devices described in this application are used to identify sequences of nucleotides incorporated into template-dependent nucleic acid sequencing reaction products synthesized by polymerase enzymes. may be used.

In certain embodiments, template-dependent nucleic acid sequencing production is performed by natural nucleic acid polymerases. In some embodiments, the polymerase is a mutant or modified variant of a native polymerase. In some embodiments, the template-dependent nucleic acid sequence product will comprise one or more nucleotide segments complementary to the template nucleic acid strand. In one aspect, the application provides a method for determining the sequence of a template (or target) nucleic acid strand by determining the sequence of its complementary nucleic acid strand.

In another aspect, the application provides a method for sequencing a target nucleic acid by sequencing a plurality of nucleic acid fragments, wherein the target nucleic acid comprises said fragments. In some embodiments, the method comprises combining multiple fragment sequences to provide a sequence or subsequence of the parental target nucleic acid. In some embodiments, the combining process is performed by computer hardware and software. The methods described herein can enable sequencing of a set of related target nucleic acids, such as an entire chromosome or genome.

During sequencing, the polymerase may be coupled (eg, attached) to the priming position of the target nucleic acid molecule. A priming position may be a primer complementary to a portion of the target nucleic acid molecule. Alternatively, the priming position is a gap or nick provided within a double-stranded segment of the target nucleic acid molecule. A gap or nick may be from 0 to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40 nucleotides in length. A nick can provide a break in one strand of a double-stranded sequence, and can provide, for example, a priming site for a polymerizing enzyme, such as a strand-displacing polymerase enzyme.

In some cases, sequencing primers can be annealed to target nucleic acid molecules that may or may not be immobilized to a solid support. Solid supports may, for example, comprise sample wells (eg, nano-apertures (reaction chambers)) on chips used for nucleic acid sequencing. In some embodiments, the sequencing primer may be immobilized to a solid support, and upon hybridization of the target nucleic acid molecule, the target nucleic acid molecule is also immobilized to the solid support. In some embodiments, the polymerase is immobilized on a solid support and the soluble primer and target nucleic acid are contacted with the polymerase. However, in some embodiments, complexes comprising polymerase, target nucleic acid, and primers are formed in solution, and the complexes are immobilized on a solid support (e.g., polymerase, primers, and/or or by immobilizing the target nucleic acid). In some embodiments, none of the components in the sample well (eg, nanoopening (reaction chamber)) are immobilized to a solid support. For example, in some embodiments, complexes comprising polymerase, target nucleic acid, and primers are formed in solution, and the complexes are not immobilized to a solid support.

Under appropriate conditions, a polymerase enzyme in contact with the annealed primer/target nucleic acid can add or incorporate one or more nucleotides into the primer, the nucleotides traversing from 5' to 3' in a template-dependent fashion. can be added to the primer with Such incorporation of nucleotides into primers (eg, by the action of a polymerase) is sometimes commonly referred to as a primer extension reaction. Each nucleotide can be detected and identified (e.g., based on its luminescence lifetime and/or other characteristics) during a nucleic acid extension reaction to determine the length of the extended primer and thus the newly synthesized nucleic acid molecule. It can be associated with a detectable tag that can be used to determine each nucleotide incorporated into a sequence. Alternatively, the sequence of the target nucleic acid molecule can be determined by sequence complementarity of newly synthesized nucleic acid molecules. In some cases, the annealing of the sequencing primer to the target nucleic acid molecule and the incorporation of nucleotides into the sequencing primer may occur under similar reaction conditions (e.g., the same or similar reaction temperature), or may occur at different temperatures. It may also occur under reaction conditions (eg, different reaction temperatures). In some embodiments, sequencing by synthetic methods presents a population of target nucleic acid molecules (e.g., copies of the target nucleic acid) and/or amplifies the target nucleic acid to achieve a population of target nucleic acids. Prepare. However, in some embodiments, sequencing-by-synthesis is used to determine the sequence of a single molecule in each reaction under evaluation (preparation of target template for sequencing requires nucleic acid amplification). and not). In some embodiments, multiple single-molecule sequencing reactions are performed in parallel (eg, on a single chip) according to one aspect of the application. For example, in some embodiments, multiple single-molecule sequencing reactions are each performed in separate reaction chambers (eg, nano-apertures, sample wells) on a single chip.

In embodiments, at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99 %, 99.999%, or 99.9999% accuracy and/or about 10 base pairs (bp), 50bp, 100bp, 200bp, 300bp, 400bp, 500bp, 1000bp, 10,000bp, 20,000bp, 30, Single nucleic acid molecules can be sequenced with high accuracy and long read lengths, such as read lengths of 000 bp, 40,000 bp, 50,000 bp, or 100,000 bp or greater. In some embodiments, the target nucleic acid molecule used for single molecule sequencing is immobilized or attached to a solid support such as the bottom or sidewall of a sample well with at least one additional component of the sequencing reaction (e.g. single-stranded target nucleic acid (e.g., deoxyribonucleic acid (DNA), DNA derivatives, ribonucleic acid (RNA ), RNA derivatives) are templates. A target nucleic acid molecule or polymerase may be attached directly or via a linker to a sample wall, such as the bottom or sidewall of a sample well. The sample wells (e.g., nanoapertures) also contain suitable buffers, cofactors, enzymes (e.g., polymerases), and luminescent tags, e.g., fluorophores, such as deoxyadenosine triphosphate (dATP) dNTPs , deoxyribonucleoside triphosphates such as deoxycytidine triphosphate (dCTP) dNTPs, deoxyguanosine triphosphate (dGTP) dNTPs, deoxyuridine triphosphate (dUTP) dNTPs, and deoxythymidine triphosphate (dTTP) dNTPs. , may contain any other reagents required for nucleic acid synthesis by a primer extension reaction. In some embodiments, each class of dNTPs (e.g., adenine-containing dNTPs (e.g., dATP ), cytosine-containing dNTPs (e.g., dCTP), guanine-containing dNTPs (e.g., dGTP), uracil-containing dNTPs (e.g., dUTP), and thymine-containing dNTPs (e.g., dTTP)) are conjugated to different luminescent tags. . Emitted light from a luminescent tag can be detected by any suitable device and/or method, including detection devices and methods described elsewhere herein, and the appropriate luminescent tag (and thus associated dNTPs). Luminescent tags can be conjugated to dNTPs at any position such that the presence of the luminescent tag does not interfere with the incorporation of the dNTP into the newly synthesized nucleic acid strand or the activity of the polymerase. In some embodiments, the luminescent tag is conjugated to the terminal phosphate (eg, gamma phosphate) of the dNTP.

In some embodiments, the single-stranded target nucleic acid template can be contacted with sequencing primers, dNTPs, polymerase, and other reagents required for nucleic acid synthesis. In some embodiments, all suitable dNTPs may contact the single-stranded target nucleic acid template at the same time (eg, all dNTPs are present at the same time) so that dNTP incorporation can occur sequentially. In other embodiments, the dNTPs may be contacted with the single-stranded target nucleic acid template sequentially, wherein the single-stranded target nucleic acid template is subjected to a washing step between contacting different dNTPs with the single-stranded target nucleic acid template. to contact each appropriate dNTP separately. Such a cycle of separately contacting the single-stranded target nucleic acid template with each dNTP followed by washing can be repeated for each consecutive base position of the single-stranded target nucleic acid template to be discriminated.

In some embodiments, the sequencing primer anneals to the single-stranded target nucleic acid template, and the polymerase serially generates dNTPs (or other deoxyribonucleoside polyphosphates) based on the single-stranded target nucleic acid template. Incorporate into the primer. The unique luminescent tag associated with each incorporated dNTP can be excited with suitable excitation light during or after incorporation of the dNTP into the primer, after which its emission is described herein. can be detected using any suitable device and/or method, including the detection devices and methods described elsewhere in . Detection of specific light emissions (eg, having specific radiative lifetimes, intensities, spectra, and/or combinations thereof) can be attributed to specific dNTPs incorporated. Sequences obtained from a panel of detected luminescent tags can then be used to determine the sequence of a single-stranded target nucleic acid template by sequence complementarity.

In this disclosure, reference is made to the dNTPs, devices, systems, and methods provided herein, but various types of nucleotides, such as ribonucleotides and deoxyribonucleotides (e.g., at least 4, 5, 6, 7 , deoxyribonucleoside polyphosphates having 8, 9, or 10 phosphate groups). Such ribonucleotides and deoxyribonucleotides may contain various types of tags (or markers) and linkers.

Nucleic Acid Sequencing Examples The following examples are intended to illustrate some of the methods, compositions, and devices described herein. All aspects of the examples are non-limiting. Figure 1 schematically illustrates the setup for single-molecule nucleic acid sequencing. 1-110 are sample wells (e.g., nano-aperture , reaction chamber). In this example, the bottom region of sample well 1-110 is illustrated as target volume (eg, excitation region) 1-120.

As described elsewhere herein, the target volume is the volume into which the excitation energy is directed. In some embodiments, the volume is a property of both the sample well volume and the coupling of excitation energy to the sample well. The target volume may be configured to limit the number of molecules or complexes localized to the target volume. In some embodiments, the target volume is configured to localize single molecules or single complexes. In some embodiments, the target volume is configured to confine a single polymerase complex. In FIG. 1, the complex containing polymerase 1-101 is confined to target volume 1-120. The complexes may optionally be immobilized by attaching them to the surface of the sample well. An exemplary process of sample well surface preparation and functionalization is illustrated in FIGS. 7-9 and discussed in greater detail elsewhere in this application. In this example, the conjugate is anchored by a linker 1-103 containing one or more biomolecules (eg biotin) suitable for attaching the linker to the polymerase 1-101.

The open volume also contains a reaction mixture with suitable solvents, buffers, and other additives necessary for the polymerase complex to synthesize nucleic acid strands. Also, the reaction mixture contains multiple types of luminescently labeled nucleotides. Each type of nucleotide is represented by the symbols *-A, @-T, $-G, #-C, where A, T, G, and C represent the nucleotide bases and the symbols *, @, $ , and # represent a unique luminescent label attached to each nucleotide via a linker '-'. In FIG. 1, the #-C nucleotide is being incorporated into complementary strand 1-102. Incorporated nucleotides are present in target volume 1-120.

Also shown conceptually in FIG. 1 are the excitation energy being applied in the vicinity of the target volume and the luminescence being emitted towards the detector by arrows. The arrows are schematic and are not intended to indicate any particular direction of excitation energy application or emission. In some embodiments, the excitation energy is a pulse of light from a light emitting source. Excitation energy may travel between the source and the vicinity of the target volume through one or more device components, such as waveguides or filters. The radiant energy may also travel between the luminescent molecules and the detector through one or more device components, such as waveguides or filters. Some emissions may radiate in vectors that are not directed to the detector (eg, to the sidewalls of the sample well) and cannot be detected.

FIG. 2 schematically illustrates the sequencing process over time in a single sample well (eg, nanoaperture). Steps A through D illustrate sample wells with polymerase complexes, as in FIG. Stage A depicts the initial state before any nucleotides are added to the primer. Step B depicts the incorporation event of a luminescent labeled nucleotide (#-C). Phase C illustrates the period between integration events. In this example, nucleotide C has been added to the primer and the label and linker previously attached to the luminescent labeled nucleotide (#-C) are cleaved. Step D illustrates the second incorporation event of a luminescent labeled nucleotide (*-A). The complementary strand after step D consists of the primer, C nucleotides and A nucleotides.

Both phases A and C illustrate the period of time before or between the incorporation event, which in this example is shown to last approximately 10 milliseconds. In steps A and C, there are no luminescence-labeled nucleotides (not depicted in FIG. 2) in the target volume because no nucleotides are being incorporated, but background luminescence and spurious signals from luminescence-labeled nucleotides not being incorporated. Luminescence may be detected. Steps B and D represent incorporation events of different nucleotides (#-C and *-A, respectively). Also, in this example, these events are shown to last approximately 10 milliseconds.

The row labeled "raw bin data" illustrates the data generated during each stage. Throughout the experimental examples, multiple pulses of light are applied near the target volume. The detector is configured to record every emitted photon received by the detector for each pulse. Emission photons are separated into one of a plurality of time bins as they are received by the detector. In this example, there are 3 of them. In some embodiments, the detector is configured with 2-16 time bins. "Raw Bin Data" records a value of 1 (shortest bar), 2 (middle bar), or 3 (longest bar), corresponding to shortest, middle, and longest bins respectively. Each bar represents the detection of emitted photons.

No photons are detected in steps A or C since there are no luminescently labeled nucleotides in the target volume. In each of stages B and D, multiple photon emission events (luminescence events, or "luminescence" as used herein) are detected during the integration event. Luminescent label '#' has a shorter luminescence lifetime than luminescent label '*'. Thus, Stage B data is illustrated as recording a lower average bin value than Stage D, which has a higher bin value.

The row labeled "processed data" shows raw data that has been processed to show the number of emitted photons (counts) at multiple time points for each pulse. Each bar corresponds to photon counts in a particular time bin, so the exemplary curve illustrating processed data corresponds to raw bin data containing more time bins than the three time bins shown in the figure. do. In this example, the data is not only processed to determine luminescence lifetime, but the data may be evaluated for other luminescence properties such as luminescence intensity or wavelength of absorbed or emitted photons. Exemplary processed data approximates the exponential decay curve characteristics of the luminescent lifetime of the luminescent label in the target volume. Luminescent label '#' has a shorter luminescence lifetime than luminescent label '*', so Stage B processed data has fewer counts at longer elapsed times, while Stage D processed data has a longer Has relatively more counts over time.

In the example experiment of Figure 2, the first two nucleotides added to the complementary strand would be identified as CA. Therefore, in the case of DNA, the sequence of the target strand immediately following the region annealed to the primer would be identified as GT. In this example, nucleotides C and A can be distinguished among multiples of C, G, T, and A based solely on emission lifetimes. In some embodiments, other properties such as emission intensity or wavelength of absorbed or emitted photons may be required to distinguish one or more particular nucleotides.

Signals emitted upon nucleotide incorporation are stored in memory and processed at a later time to determine the sequence of the target nucleic acid template. This involves comparing the signal to a reference signal to determine the identity of incorporated nucleotides as a function of time. Alternatively or additionally, signals emitted upon nucleotide incorporation can be collected and processed in real time (e.g., upon nucleotide incorporation) to determine the sequence of the target nucleic acid template in real time. is.

The term "nucleic acid" as used herein generally refers to molecules comprising one or more nucleic acid subunits. Nucleic acids can include one or more subunits selected from adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. can. In some examples, the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or derivatives thereof. Nucleic acids may be single-stranded or double-stranded. Nucleic acids may be circular.

The term "nucleotide" as used herein generally refers to a nucleic acid subunit that may contain A, C, G, T, or U, or variants or analogs thereof. A nucleotide may include any subunit that is capable of being incorporated into a growing nucleic acid chain. Such subunits may be A, C, G, T, or U, or one or more complementary A, C, G, T or U specific or purine (e.g. , A or G, or variants or analogs thereof) or any other subunit complementary to a pyrimidine (e.g., C, T, or U, or variants or analogs thereof). . Subunits allow distinguishing between individual nucleobases or groups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, or their uracil equivalents) can do.

Nucleotides generally include a nucleoside and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphate ( _PO3 ) groups. A nucleotide may include a nucleobase, a pentose sugar (either ribose or deoxyribose), and one or more phosphate groups. Ribonucleotides are nucleotides in which the sugar is ribose. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose. Nucleotides may be nucleoside monophosphates or nucleoside polyphosphates. Nucleotides include, e.g., deoxyadenosine triphosphate (dATP) dNTPs, deoxycytidine triphosphate (dCTP) dNTPs, deoxyguanosine triphosphate (dGTP), including detectable tags such as luminescent tags or markers (e.g., fluorophores) ) deoxyribonucleoside polyphosphates, such as deoxyribonucleoside triphosphates, which can be selected from dNTPs, deoxyuridine triphosphate (dUTP) dNTPs, and deoxythymidine triphosphate (dTTP) dNTPs.

Nucleoside polyphosphates may have "n" phosphate groups, where "n" is a number of 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. Examples of nucleoside polyphosphates include nucleoside diphosphates and nucleoside triphosphates. A nucleotide may be a terminal phosphate-labeled nucleoside, such as a terminal phosphate-labeled nucleoside polyphosphate. Such labels may be luminescent (eg, fluorescent or chemiluminescent) labels, fluorogenic labels, colored labels, chromogenic labels, mass tags, electrostatic labels, or electrochemical labels. A label (or marker) may be coupled to the terminal phosphate via a linker. The linker may comprise, for example, at least one or more hydroxyl, sulfhydryl, amino, or haloalkyl groups, which are used, for example, for phosphate ester linkages at terminal phosphates of natural or modified nucleotides, It may be suitable for forming thioester, phosphoramidate, or alkylphosphonate linkages. The linker may be cleavable, such as by a polymerase, to separate the label from the terminal phosphate. Examples of nucleotides and linkers are provided in US Pat. No. 7,041,812, which is hereby incorporated by reference in its entirety.

Nucleotides (eg, nucleotide polyphosphates) may contain methylated nucleobases. For example, a methylated nucleotide comprises one or more methyl groups attached to a nucleobase (e.g., attached directly to a nucleobase ring, attached to a substituent of a nucleobase ring). It may be a nucleotide. Exemplary methylated nucleobases include 1-methylthymine, 1-methyluracil, 3-methyluracil, 3-methylcytosine, 5-methylcytosine, 1-methyladenine, 2-methyladenine, 7-methyladenine, N6 -methyladenine, N6,N6-dimethyladenine, 1-methylguanine, 7-methylguanine, N2-methylguanine, and N2,N2-dimethylguanine.

The term "primer," as used herein, is generally a nucleic acid that may contain a sequence that includes A, C, G, T, and/or U, or variants or analogs thereof. It refers to a molecule (eg, an oligonucleotide). A primer may be a synthetic oligonucleotide comprising DNA, RNA, PNA, or variants or analogs thereof. The primer may be designed such that its nucleotide sequence is complementary to the target strand, or the primer may contain random nucleotide sequences. In some embodiments, primers may include tails (eg, poly-A tails, index adapters, molecular barcodes, etc.). In some embodiments, the primer is 5-15 bases, 10-20 bases, 15-25 bases, 20-30 bases, 25-35 bases, 30-40 bases bases, 35-45 bases, 40-50 bases, 45-55 bases, 50-60 bases, 55-65 bases, 60-70 bases, 65-75 bases , 70-80 bases, 75-85 bases, 80-90 bases, 85-95 bases, 90-100 bases, 95-105 bases, 100-150 bases, It may contain 125-175 bases, 150-200 bases, or more than 200 bases.

Luminescent Properties As described herein, luminescent molecules are molecules that can absorb one or more photons and then emit one or more photons after one or more lapses of time. is. The luminescence of a molecule is described by several parameters including, but not limited to, luminescence lifetime, absorption spectrum, emission spectrum, luminescence quantum yield, and luminescence intensity. The terms "absorption" and "excitation" are used synonymously throughout this application. A typical luminescent molecule can absorb multiple wavelengths of light or undergo excitation with multiple wavelengths of light. Excitation at certain wavelengths or within certain spectral ranges may be mitigated by luminescence emission events, while excitation at certain other wavelengths or within spectral ranges may not be mitigated by luminescence emission events. In some embodiments, a luminescent molecule is appropriately excited to emit light only at a single wavelength or within a single spectral range. In some embodiments, the luminescent molecule emits light upon proper excitation at two or more wavelengths or within two or more spectral ranges. In some embodiments, molecules are identified by measuring the wavelength of the excitation photon or the absorption spectrum.

Emission photons from a luminescence emission event will emit at wavelengths within the spectral range of possible wavelengths. Typically, the emission photons have a longer wavelength (eg, have less energy or are red-shifted) compared to the wavelength of the excitation photons. In some embodiments, molecules are identified by measuring the wavelength of emitted photons. In some embodiments, molecules are identified by measuring the wavelengths of multiple emitted photons. In some embodiments, molecules are identified by measuring their emission spectra.

Luminescence lifetime refers to the elapsed time between an excitation event and an emission event. In some embodiments, the luminescence lifetime is expressed as a constant in the exponential decay equation. In some embodiments, the excitation energy is applied by one or more pulse events, and the elapsed time is the time between the pulse and the subsequent emission event.

"Determining the luminescence lifetime" of a molecule can be performed in any suitable manner (e.g., by measuring the lifetime using a suitable technique, or determining the time-dependent characteristics of the emission by doing). In some embodiments, determining the luminescent lifetime of a molecule comprises determining lifetimes for one or more molecules (eg, different luminescent labeled nucleotides in a sequencing reaction). In some embodiments, determining the luminescence lifetime of the molecule comprises determining the lifetime for a reference entity. In some embodiments, determining the emission lifetime of a molecule comprises measuring lifetime (eg, fluorescence lifetime). In some embodiments, determining the luminescence lifetime of the molecule comprises determining one or more temporal features indicative of lifetime. In some embodiments, the luminescence lifetime of a molecule can be determined based on the distribution of multiple emission events that occur over one or more time-gated windows for an excitation pulse (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more radiation events). For example, the emission lifetime of a single molecule can be distinguished from multiple molecules with different emission lifetimes based on the distribution of photon arrival times measured with respect to the excitation pulse.

The emission lifetime of a single molecule indicates the timing of photons emitted after the single molecule reaches an excited state, and the single molecule can be discriminated by information indicating the timing of the photons. should be understood. Some embodiments may include distinguishing a molecule from a plurality of molecules based on the emission lifetime of the molecule by measuring the time associated with the photon emitted by the molecule. The time distribution can provide an indication of luminescence lifetime that can be determined from the distribution. In some embodiments, a single molecule can be distinguished from a plurality of molecules based on the distribution of time, such as by comparing the distribution of time to a reference distribution corresponding to known molecules. In some embodiments, the luminescence lifetime value is determined from the time distribution.

Luminescence quantum yield refers to the fraction of excitation events at a given wavelength or within a given spectral range that lead to emission events, and is typically less than one. In some embodiments, the emission quantum yield of the molecules described herein is 0 to about 0.001, about 0.001 to about 0.01, about 0.01 to about 0.1, about 0.1 to about 0.5, about 0.5 to 0.9, or about 0.9 to 1. In some embodiments, molecules are identified by determining or estimating their emission quantum yield.

As used herein, single-molecule luminescence intensity refers to the number of emitted photons per unit time emitted by a molecule that has been excited by the application of a pulsed excitation energy. In some embodiments, luminescence intensity is the detected number of emitted photons per unit time emitted by a molecule that has been excited by applying a pulsed excitation energy and detected by a particular sensor or set of sensors. Point.

Emission lifetimes, emission quantum yields, and emission intensities can vary for a given molecule under different conditions. In some embodiments, the luminescence lifetime, luminescence quantum yield, or luminescence intensity observed for a single molecule will be different than for an assembly of molecules. In some embodiments, the luminescence lifetime, luminescence quantum yield, or luminescence intensity observed for molecules that are confined to a sample well (e.g., nanoaperture) is different than for molecules that are not confined to a sample well. would be different. In some embodiments, a luminescent label or molecule attached to another molecule has a different emission lifetime, emission quantum yield, or emission intensity than a luminescent label or molecule not attached to another molecule. right. In some embodiments, a molecule that interacts with a macromolecular complex (e.g., a protein complex (e.g., a nucleic acid polymerase)) has a different emission lifetime, emission quantum yield, or emission quantum yield than a molecule that does not interact with the macromolecular complex. or will have an emission intensity.

In certain embodiments, the luminescent molecules described in this application absorb one photon and emit one photon after some elapsed time. In some embodiments, the luminescent lifetime of a molecule can be determined or estimated by measuring elapsed time. In some embodiments, the luminescence lifetime of a molecule can be determined or estimated by measuring multiple elapsed times of multiple pulse events and radiation events. In some embodiments, the luminescence lifetime of a molecule can be distinguished from the luminescence lifetimes of multiple types of molecules by measuring elapsed time. In some embodiments, the luminescence lifetime of a molecule can be distinguished from the luminescence lifetimes of multiple types of molecules by measuring multiple elapsed times of multiple pulse events and radiation events. In certain embodiments, a molecule is identified or distinguished from multiple types of molecules by determining or estimating the emission lifetime of the molecule. In certain embodiments, a molecule is identified or distinguished from multiple types of molecules by distinguishing the emission lifetime of the molecule from multiple emission lifetimes of multiple types of molecules.

In some embodiments, the luminescence emission event is fluorescence. In some embodiments, the luminescence emission event is phosphorescence. As used herein, the term "luminescence" encompasses all luminescent events, including both fluorescence and phosphorescence.

In one aspect, the present application provides a method for determining the luminescence lifetime of a single luminescent molecule comprising the steps of providing the luminescent molecule in a target volume, multiple pulses of excitation energy in the vicinity of the target volume and detecting a plurality of emissions from the luminescent molecule. In some embodiments, the method further comprises evaluating a distribution of multiple elapsed times between each pair of pulses and emissions. In some embodiments, the method further comprises immobilizing a single luminescent molecule in the target volume.

In another aspect, the present application provides a method for determining the luminescence lifetime of a plurality of molecules, comprising: providing a plurality of luminescent molecules in a target volume; A method is provided comprising pulsing and detecting a plurality of emissions from a luminescent molecule. In some embodiments, the method further comprises evaluating a distribution of multiple elapsed times between each pair of pulses and emissions. In some embodiments, the method further comprises immobilizing a plurality of luminescent molecules in the target volume. In some embodiments, a plurality consists of 2 to about 10 molecules, about 10 to about 100 molecules, or about 100 to 1000 molecules. In some embodiments, the plurality is from about 1000 to about 10 ⁶ molecules, from about 10 ⁶ to about 10 ⁹ molecules, from about 10 ⁹ to about 10 ¹² molecules, from about 10 ¹² to about 10 ¹⁵ molecules, or from about 10 ¹⁵ to about 10 ¹⁸ molecules. In some embodiments, the multiple molecules are all of the same type of molecule.

Exemplary decay profiles 3-1 of four luminescent molecules with different emission lifetimes (longest to shortest, top to bottom) are displayed in FIG. The intensity of luminescence from a sample containing a large number of molecules is sometimes called the amplitude. After the initial excitation, the amplitude decreases exponentially with time according to the emission lifetime. Alternatively, amplitude may refer to the number or count of emissions detected after some elapsed time after multiple pulses of excitation energy for a single molecule, for example. For four luminescent molecules with different emission lifetimes (shortest to longest, top to bottom), the normalized cumulative distribution function 3-2 corresponds to 3-1. The CDF can represent the normalized probability that the emission amplitude reaches zero over time according to the emission lifetime after initial excitation (eg, the cumulative probability of all excited molecules emitting light). Alternatively, the CDF can represent the normalized probability of a single molecule emitting luminescence at some elapsed time after a single pulse of excitation energy or after each of multiple pulses.

In one aspect, the present application provides a method for determining the emission intensity of a single luminescent molecule comprising the steps of: providing the luminescent molecule in a target volume; multiple pulses of excitation energy in the vicinity of the target volume; and detecting a plurality of emissions from the luminescent molecule. In some embodiments, the method further comprises determining the number of multiple detected emissions per unit time. In some embodiments, the method further comprises immobilizing a single luminescent molecule in the target volume.

In another aspect, the present application provides a method for determining the luminescence intensity of a plurality of molecules, comprising: providing a plurality of luminescent molecules in a target volume; A method is provided comprising pulsing and detecting a plurality of emissions from a luminescent molecule. In some embodiments, the method further comprises determining the number of multiple detected emissions per unit time. In some embodiments, the method further comprises immobilizing a plurality of luminescent molecules in the target volume. In some embodiments, a plurality consists of 2 to about 10 molecules, about 10 to about 100 molecules, or about 100 to 1000 molecules. In some embodiments, the plurality is from about 1000 to about 10 ⁶ molecules, from about 10 ⁶ to about 10 ⁹ molecules, from about 10 ⁹ to about 10 ¹² molecules, from about 10 ¹² to about 10 ¹⁵ molecules, or from about 10 ¹⁵ to about 10 ¹⁸ molecules. In some embodiments, the multiple molecules are all of the same type of molecule.

Excitation Energy In one aspect of the methods described herein, one or more excitation energies are used to excite the luminescent label of the molecule to be identified or distinguished. In some embodiments, the excitation energy is within the visible spectrum. In some embodiments the excitation energy is in the ultraviolet spectrum. In some embodiments the excitation energy is in the infrared spectrum. In some embodiments, one excitation energy is used to excite the luminescent labeled molecule. In some embodiments, two excitation energies are used to excite the luminescent labeled molecule. In some embodiments, three or more excitation energies are used to excite the luminescent labeled molecule. In some embodiments, each luminescent labeled molecule is excited by only one of the applied excitation energies. In some embodiments, the luminescent labeled molecule is excited by two or more of the applied excitation energies. In some embodiments, the excitation energy may be monochromatic or confined to a spectral range. In some embodiments, the spectral range ranges from about 0.1 nm to about 1 nm, from about 1 nm to about 2 nm, or from about 2 nm to about 5 nm. In some embodiments, the spectral range ranges from about 5 nm to about 10 nm, from about 10 nm to about 50 nm, or from about 50 nm to about 100 nm.

In some embodiments, the excitation energy is applied as pulses of light. In some embodiments, the excitation energy is applied as multiple pulses of light. In some embodiments, two or more excitation energies are used to excite the luminescent labeled molecule. In some embodiments, each excitation energy is applied simultaneously (eg, in each pulse). In some embodiments, each excitation energy is applied at different times (eg, in separate pulses of each energy). Different excitation energies can be applied in any pattern sufficient to allow detection of luminescence from the target molecule. In some embodiments, two excitation energies are applied in each pulse. In some embodiments, the first excitation energy and the second excitation energy are applied in alternating pulses. In some embodiments, the first excitation energy is applied in a series of sequential pulses and the second excitation energy is applied in a subsequent series of sequential pulses or in a series of such alternating patterns.

In some embodiments, the frequency of the pulses of light is selected based on the luminescent properties of the luminescent label molecule. In some embodiments, the frequency of the pulses of light is selected based on the luminescence properties of the plurality of luminescently labeled nucleotides. In some embodiments, the frequency of the pulses of light is selected based on the luminescence lifetimes of the plurality of luminescently labeled nucleotides. In some embodiments, the frequency is selected such that the interval between pulses is longer than the luminescent lifetime of one or more luminescently labeled nucleotides. In some embodiments, the frequency is selected based on the longest luminescent lifetime of multiple luminescently labeled nucleotides. For example, if the emission lifetimes of four luminescently labeled nucleotides are 0.25, 0.5, 1.0, and 1.5 ns, the frequency of the pulses of light will be greater than 1.5 ns between pulses. may be selected as In some embodiments, the spacing is about 2 to about 10 times, about 10 to about 100 times, or about 100 to about 10 times longer than the emission lifetime of one or more luminescent label molecules being excited. Only 1000 times longer. In some embodiments, the interval is about 10 times longer than the emission lifetime of the one or more luminescent labeled molecules being excited. In some embodiments, the interval is about 0.01 ns to about 0.1 ns, about 1 ns to about 5 ns, about 5 ns to about 15 ns, about 15 ns to about 25 ns, or about 25 ns to about 50 ns. In some embodiments, the interval is 50%, 75%, 90%, 50%, 75%, 90%, Selected to be 95% or 99%.

In some embodiments, when multiple excitation energies are present, the frequency of each excitation energy pulse is the same. In some embodiments, when multiple excitation energies are present, the frequency of each excitation energy pulse is different. For example, if a red laser is used to excite luminescent molecules with lifetimes of 0.2 ns and 0.5 ns and a green laser is used to excite luminescent molecules with lifetimes of 5 ns and 7 ns, then each red The interval after a laser pulse may be shorter (eg 5 ns) than the interval after each green laser pulse (eg 20 ns).

In some embodiments, the frequency of pulsed excitation energy is selected based on the chemical process being monitored. For sequencing reactions, the frequency can be selected such that several pulses are applied to allow detection of a sufficient number of emitted photons to be detected. A sufficient number in the context of photon detection refers to the number of photons required to distinguish or discriminate a luminescence-labeled nucleotide from a plurality of luminescence-labeled nucleotides. For example, a DNA polymerase can incorporate an additional nucleotide on average every 20 milliseconds. The time for a luminescent-labeled nucleotide to interact with the complex may be about 10 ms, and the time between cleavage of the luminescent marker and the start of interaction of the next luminescent-labeled nucleotide is about 10 ms. may be The frequency of the pulsed excitation energy can then be selected to apply sufficient pulses for 10 milliseconds such that a sufficient number of emitted photons are detected in 10 milliseconds during incorporation of the luminescently labeled nucleotide. It is possible. For example, at a frequency of 100 MHz, a pulse of 10 milliseconds (the approximate length of the built-in event) would be 1 million times. If 0.1% of these pulses resulted in photon detection, there would be 1,000 luminescence data points that could be analyzed to determine the identity of the luminescence-labeled nucleotide during incorporation. Any of the above values are non-limiting. In some embodiments, an integration event may take between 1 ms and 20 ms, between 20 ms and 100 ms, or between 100 ms and 500 ms. In some embodiments in which multiple excitation energies are applied in temporally separated pulses, the luminescently labeled nucleotide may be excited only during a portion of the pulse. In some embodiments, the frequency and pattern of the plurality of excitation energy pulses is such that the number of pulses excites any one of the plurality of luminescently labeled nucleotides to produce a sufficient number of emitted photons to be detected. It is chosen to be sufficient to allow detection.

In some embodiments, the frequency of pulses is from about 1 MHz to about 10 MHz. In some embodiments, the frequency of pulses is from about 10 MHz to about 100 MHz. In some embodiments, the frequency of pulses is from about 100 MHz to about 1 GHz. In some embodiments, the frequency of pulses is from about 50 MHz to about 200 MHz. In some embodiments, the frequency of pulses is about 100 MHz. In some embodiments, the frequencies are stochastic.

In some embodiments, the excitation energy is between about 500 nm and about 700 nm. In some embodiments, the excitation energy is from about 500 nm to about 600 nm, or from about 600 nm to about 700 nm. In some embodiments, the excitation energy is from about 500 nm to about 550 nm, from about 550 nm to about 600 nm, from about 600 nm to about 650 nm, or from about 650 nm to about 700 nm.

In some embodiments, the methods described herein comprise applying two excitation energies. In some embodiments, the two excitation energies are about 5 nm to about 20 nm, about 20 nm to about 40 nm, about 40 nm to about 60 nm, about 60 nm to about 80 nm, about 80 nm to about 100 nm, about 100 nm to about 150 nm, about separated by 150 nm to about 200 nm, about 200 nm to about 400 nm, or at least about 400 nm. In some embodiments, the two excitation energies are separated by about 20 nm to about 80 nm or about 80 nm to about 160 nm.

Where the excitation energy is referred to as being in a particular range, the excitation energy may include a single wavelength such that the wavelength is between or at the endpoints of the range, or the excitation energy may be , may include a spectrum of wavelengths with maxima such that the maxima are between ranges or at the endpoints of ranges.

In some embodiments, the first excitation energy ranges from 450 nm to 500 nm and the second excitation energy ranges from 500 nm to 550 nm, 550 nm to 600 nm, 600 nm to 650 nm, or 650 nm to 700 nm. In some embodiments, the first excitation energy ranges from 500 nm to 550 nm and the second excitation energy ranges from 450 nm to 500 nm, 550 nm to 600 nm, 600 nm to 650 nm, or 650 nm to 700 nm. In some embodiments, the first excitation energy ranges from 550 nm to 600 nm and the second excitation energy ranges from 450 nm to 500 nm, 500 nm to 550 nm, 600 nm to 650 nm, or 650 nm to 700 nm. In some embodiments, the first excitation energy ranges from 600 nm to 650 nm and the second excitation energy ranges from 450 nm to 500 nm, 500 nm to 550 nm, 550 nm to 600 nm, or 650 nm to 700 nm. In some embodiments, the first excitation energy ranges from 650 nm to 700 nm and the second excitation energy ranges from 450 nm to 500 nm, 500 nm to 550 nm, 550 nm to 600 nm, or 600 nm to 650 nm.

In some embodiments, the first excitation energy ranges from 450 nm to 500 nm and the second excitation energy ranges from 500 nm to 550 nm. In some embodiments, the first excitation energy ranges from 450 nm to 500 nm and the second excitation energy ranges from 550 nm to 600 nm. In some embodiments, the first excitation energy ranges from 450 nm to 500 nm and the second excitation energy ranges from 600 nm to 670 nm. In some embodiments, the first excitation energy ranges from 500 nm to 550 nm and the second excitation energy ranges from 550 nm to 600 nm. In some embodiments, the first excitation energy ranges from 500 nm to 550 nm and the second excitation energy ranges from 600 nm to 670 nm. In some embodiments, the first excitation energy ranges from 550 nm to 600 nm and the second excitation energy ranges from 600 nm to 670 nm. In some embodiments, the first excitation energy ranges from 470 nm to 510 nm and the second excitation energy ranges from 510 nm to 550 nm. In one embodiment, the first excitation energy ranges from 470 nm to 510 nm and the second excitation energy ranges from 550 nm to 580 nm. In one embodiment, the first excitation energy ranges from 470 nm to 510 nm and the second excitation energy ranges from 580 nm to 620 nm. In some embodiments, the first excitation energy ranges from 470 nm to 510 nm and the second excitation energy ranges from 620 nm to 670 nm. In some embodiments, the first excitation energy ranges from 510 nm to 550 nm and the second excitation energy ranges from 550 nm to 580 nm. In some embodiments, the first excitation energy ranges from 510 nm to 550 nm and the second excitation energy ranges from 580 nm to 620 nm. In some embodiments, the first excitation energy ranges from 510 nm to 550 nm and the second excitation energy ranges from 620 nm to 670 nm. In some embodiments, the first excitation energy ranges from 550 nm to 580 nm and the second excitation energy ranges from 580 nm to 620 nm. In one embodiment, the first excitation energy ranges from 550 nm to 580 nm and the second excitation energy ranges from 620 nm to 670 nm. In some embodiments, the first excitation energy ranges from 580 nm to 620 nm and the second excitation energy ranges from 620 nm to 670 nm.

Certain embodiments of excitation energy sources and devices for applying pulses of excitation energy to a target volume are described elsewhere herein.
Luminescently Labeled Nucleotides In one aspect, the methods and compositions described herein comprise one or more luminescently labeled nucleotides. In certain embodiments, one or more nucleotides comprises a deoxyribose nucleoside. In some embodiments, all nucleotides include deoxyribose nucleosides. In certain embodiments, one or more nucleotides comprises a ribose nucleoside. In some embodiments, all nucleotides include ribose nucleosides. In some embodiments, one or more nucleotides comprise a modified ribose sugar or ribose analogue (eg, locked nucleic acid). In some embodiments, one or more nucleotides include natural bases (eg, cytosine, guanine, adenine, thymine, uracil). In some embodiments, one or more nucleotides comprise a derivative or analogue of cytosine, guanine, adenine, thymine, or uracil.

In some embodiments, the method comprises exposing the polymerase complex to a plurality of luminescently labeled nucleotides. In some embodiments, the composition or device comprises a reaction mixture comprising a plurality of luminescently labeled nucleotides. In some embodiments the plurality of nucleotides comprises four types of nucleotides. In some embodiments, each of the four types of nucleotides includes one of cytosine, guanine, adenine, and thymine. In some embodiments, each of the four types of nucleotides includes one of cytosine, guanine, adenine, and uracil.

In certain embodiments, the concentration of each type of luminescently labeled nucleotide in the reaction mixture is about 50 nM to about 200 nM, about 200 nM to about 500 nM, about 500 nM to about 1 μM, about 1 μM to about 50 μM, or about 50 μM to 250 μM. . In some embodiments, the concentration of each type of luminescently labeled nucleotide in the reaction mixture is from about 250 nM to about 2 μM. In some embodiments, the concentration of each type of luminescently labeled nucleotide in the reaction mixture is about 1 μM.

In certain embodiments, the reaction mixture contains additional reagents used in the sequencing reaction. In some embodiments, the reaction mixture includes a buffer. In some embodiments, the buffering agent comprises 3-(N-morpholino)propanesulfonic acid (MOPS). In some embodiments, buffering agents are present at concentrations from about 1 mM to about 100 mM. In some embodiments, the concentration of MOPS is about 50 mM. In some embodiments, the reaction mixture includes one or more salts. In some embodiments the salt comprises potassium acetate. In some embodiments, the concentration of potassium acetate is about 140 mM. In some embodiments, the salt is present at a concentration of about 1 mM to about 200 mM. In some embodiments, the reaction mixture includes a magnesium salt (eg, magnesium acetate). In some embodiments, the concentration of magnesium acetate is about 20 mM. In some embodiments, the magnesium salt is present at a concentration of about 1 mM to about 50 mM. In some embodiments, the reaction mixture includes a reducing agent. In some embodiments, the reducing agent is dithiothreitol (DTT). In some embodiments, the reducing agent is present at a concentration of about 1 mM to about 50 mM. In some embodiments, the concentration of DTT is about 5 mM. In some embodiments, the reaction mixture includes one or more light stabilizers. In some embodiments, the reaction mixture includes an antioxidant, oxygen scavenger, or triplet state quencher. In some embodiments, the light stabilizer comprises protocatechuic acid (PCA). In some embodiments, the light stabilizer comprises 4-nitrobenzyl alcohol (NBA). In some embodiments, the photostabilizer is present at a concentration of about 0.1 mM to about 20 mM. In some embodiments, the concentration of PCA is about 3 mM. In some embodiments, the concentration of NBA is about 3 mM. Mixtures with light stabilizers (eg, PCA) may also include an enzyme (eg, protocatechuate dioxygenase (PCD)) to regenerate the light stabilizer. In some embodiments, the concentration of PCD is about 0.3 mM.

The present application contemplates various methods for distinguishing nucleotides from multiple nucleotides. In some embodiments, each of the luminescently labeled nucleotides has a different luminescent lifetime. In certain embodiments, two or more of the luminescently labeled nucleotides have the same or substantially the same luminescent lifetime (eg, lifetimes that cannot be determined by the methods or devices described above).

In certain embodiments, each of the luminescently labeled nucleotides absorbs excitation energies in different spectral ranges. In some embodiments, two of the luminescently labeled nucleotides absorb excitation energies in the same spectral range. In some embodiments, three of the luminescently labeled nucleotides absorb excitation energies in the same spectral range. In some embodiments, four or more of the luminescently labeled nucleotides absorb excitation energies in the same spectral range. In certain embodiments, two of the luminescently labeled nucleotides absorb excitation energies in different spectral ranges. In some embodiments, three of the luminescently labeled nucleotides absorb excitation energies in different spectral ranges. In some embodiments, four or more of the luminescently labeled nucleotides absorb excitation energies in different spectral ranges.

In some embodiments, each of the luminescently labeled nucleotides emits photons in different spectral ranges. In some embodiments, two of the luminescently labeled nucleotides emit photons in the same spectral range. In some embodiments, three of the luminescently labeled nucleotides emit photons in the same spectral range. In some embodiments, four or more of the luminescently labeled nucleotides emit photons in the same spectral range. In some embodiments, two of the luminescently labeled nucleotides emit photons in different spectral ranges. In some embodiments, three of the luminescently labeled nucleotides emit photons in different spectral ranges. In some embodiments, four or more of the luminescently labeled nucleotides emit photons in different spectral ranges.

In some embodiments, each of the four luminescently labeled nucleotides has a different luminescent lifetime. In certain embodiments, two or more luminescently labeled nucleotides have different luminescent lifetimes and absorb and/or emit photons in a first spectral range, and one or more luminescently labeled nucleotides are in a second spectral range. Absorbs and/or emits a range of photons. In some embodiments, each of the three luminescent-labeled nucleotides has a different emission lifetime and emits luminescence in a first spectral range, and the fourth luminescent-labeled nucleotide emits photons in a second spectral range. Absorb and/or emit. In some embodiments, each of the two luminescent-labeled nucleotides has a different emission lifetime and emits in the first spectral range, and the third and fourth luminescent-labeled nucleotides each have a different emission lifetime. and emits emission in the second spectral range.

In some embodiments, each of the four luminescently labeled nucleotides has a different emission intensity. In certain embodiments, two or more luminescently labeled nucleotides have different emission intensities and emit luminescence in a first spectral range, and one or more luminescently labeled nucleotides emit photons in a second spectral range. Absorb and/or emit. In some embodiments, each of the three luminescently labeled nucleotides has a different emission intensity and emits luminescence in a first spectral range, and the fourth luminescently labeled nucleotide emits photons in a second spectral range. Absorb and/or emit. In some embodiments, each of the two luminescently labeled nucleotides has a different emission intensity and emits in the first spectral range, and the third and fourth luminescently labeled nucleotides each have a different emission intensity. and emits emission in the second spectral range.

In some embodiments, each of the four luminescently labeled nucleotides has a different luminescent lifetime or intensity. In certain embodiments, two or more luminescent-labeled nucleotides have different emission lifetimes or luminescence intensities and emit light in a first spectral range, and one or more luminescent-labeled nucleotides emit in a second spectral range. photons are absorbed and/or emitted. In some embodiments, each of the three luminescent-labeled nucleotides has a different emission lifetime or emission intensity and emits in a first spectral range, and the fourth luminescent-labeled nucleotide emits in a second spectral range. photons are absorbed and/or emitted. In some embodiments, each of the two luminescent-labeled nucleotides has a different emission lifetime or emission intensity and emits light in the first spectral range, and the third and fourth luminescent-labeled nucleotides each have a different It has an emission lifetime or emission intensity and emits emission in a second spectral range.

In certain embodiments, two or more luminescently labeled nucleotides have different luminescent lifetimes and absorb excitation energy in a first spectral range, and one or more luminescently labeled nucleotides absorb excitation energy in a second spectral range. Absorb energy. In some embodiments, each of the three luminescently labeled nucleotides has a different luminescent lifetime and absorbs excitation energy in a first spectral range, and the fourth luminescently labeled nucleotide absorbs excitation energy in a second spectral range. absorb. In some embodiments, each of the two luminescent-labeled nucleotides has a different emission lifetime and absorbs excitation energy in the first spectral range, and the third and fourth luminescent-labeled nucleotides each have a different emission lifetime. and absorbs excitation energy in the second spectral range.

In certain embodiments, two or more luminescent-labeled nucleotides have different luminescence lifetimes or luminescence intensities and absorb excitation energy in a first spectral range, and one or more luminescent-labeled nucleotides absorbs excitation energy in a second spectral range. Absorbs a range of excitation energies. In some embodiments, each of the three luminescently labeled nucleotides has a different emission lifetime or emission intensity and absorbs excitation energy in a first spectral range, and the fourth luminescently labeled nucleotide is in a second spectral range. absorbs the excitation energy of In some embodiments, each of the two luminescent-labeled nucleotides has a different emission lifetime or emission intensity and absorbs excitation energy in the first spectral range, and the third and fourth luminescent-labeled nucleotides each have a different It has a luminescence lifetime or luminescence intensity and absorbs excitation energy in a second spectral range.

During sequencing, the method for distinguishing nucleotides may vary between different base pairs of a sequence. In some embodiments, two types of nucleotides may be labeled to absorb the first excitation energy, and the two types of nucleotides (e.g., A, G) are labeled based on different emission intensities. Discriminated, two additional types of nucleotides (e.g., C, T) may be labeled to absorb the second excitation energy, and the two additional types of nucleotides have different emission lifetimes. determined based on For such embodiments, during sequencing, certain segments of the sequence may be determined based on luminescence intensity alone (e.g., segments incorporating only A and G) and other segments of the sequence may be determined based solely on the emission lifetime (eg, segments incorporating only C and T). In some embodiments, the 2-4 luminescently labeled nucleotides will be distinguished based on luminescent lifetime. In some embodiments, the 2-4 luminescently labeled nucleotides are distinguished based on luminescence intensity. In some embodiments, the 2-4 luminescently labeled nucleotides are distinguished based on luminescence lifetime and luminescence intensity.

FIG. 4 displays the luminescence lifetime 4-1 of an exemplary luminescent-labeled nucleotide and the luminescence intensity 4-2 of the same exemplary nucleotide. For example, the fourth row presents data for deoxythymidine hexaphosphate (dT6P) nucleotides linked to the fluorophore Alexa Fluor® 555 (AF555). This luminescent labeled nucleotide has a lifetime of approximately 0.25 ns and exhibits an emission intensity of approximately 20000 counts/second. The observed luminescence lifetime and luminescence intensity of any luminescence-labeled nucleotide is generally different for the nucleotide under incorporation conditions compared to other more typical conditions such as those of 4-1 and 4-2. (eg, single-molecule complexes in nanoapertures).

Luminescence Detection In one aspect of the methods described herein, the emitted photon (luminescence) or plurality of emitted photons are detected by one or more sensors. The plurality of luminescently labeled molecules or nucleotides may each of the molecules emit photons in a single spectral range, or a portion of the molecules may emit photons in a first spectral range; Another portion may emit photons in a second spectral range. In some embodiments, emitted photons are detected by a single sensor. In some embodiments, the emitted photons are detected by multiple sensors. In some embodiments, photons emitted in the first spectral range are detected by a first sensor and photons emitted in the second spectral range are detected by a second sensor. In some embodiments, photons emitted in each of the multiple spectral ranges are detected by different sensors.

In some embodiments, each sensor is configured to assign a time bin to the emitted photon based on the elapsed time between the excitation energy and the emitted photon. In some embodiments, photons emitted after a shorter elapsed time will be attributed to earlier time bins, and photons emitted after a longer duration will be attributed to later time bins.

In some embodiments, multiple pulses of excitation energy are applied in the vicinity of the target volume and multiple photons are detected, which may include photon emission events. In some embodiments, multiple luminescence (eg, photon emission events) correspond to the incorporation of luminescence-labeled nucleotides into the nucleic acid product. In some embodiments, incorporation of the luminescent-labeled nucleotide continues for about 1 ms to about 5 ms, about 5 ms to about 20 ms, about 20 ms to about 100 ms, or about 100 ms to about 500 ms. In some embodiments, about 10 to about 100, about 100 to about 1000, about 1000 to about 10,000, or about 10,000 to about 100,000 luminescences are detected during incorporation of the luminescent-labeled nucleotide.

In some embodiments, no luminescence is detected when the luminescent-labeled nucleotide is not being incorporated. In some embodiments, a luminescent background is present. In some embodiments, pseudoluminescence is detected when the luminescence-labeled nucleotide is not being incorporated. During the pulse of excitation energy, one or more luminescently labeled nucleotides are present in the target volume (e.g., diffuse into the target volume or interact with the polymerase but are not incorporated), but sequencing Such spurious luminescence may occur when the reaction is not incorporating. In some embodiments, the multiple emissions detected from the luminescent-labeled nucleotides present in the target volume but not being incorporated are lower than the multiple emissions from the luminescent-labeled nucleotides (e.g., 10-fold, 100-fold , x1000, x10000).

In some embodiments, each of the plurality of detected emissions corresponding to incorporation of a luminescently labeled nucleotide is assigned to a time bin based on the elapsed time between the pulse and the emitted photon. Such multiple occurrences of built-in events are referred to herein as "bursts." In some embodiments, a burst refers to a series of signals (eg, measurements) that exceed a baseline (eg, noise threshold). In this case, the signal corresponds to multiple emission events that occur when luminescently labeled nucleotides are present within the excitation region. In some embodiments, bursts are separated from previous and/or subsequent bursts by the time interval of the signal representing the baseline. In some embodiments, bursts are analyzed by determining luminescence lifetimes based on multiple elapsed times. In some embodiments, bursts are analyzed by determining emission intensity based on the number of emissions detected per unit time. In some embodiments, bursts are analyzed by determining the spectral range of detected emissions. In some embodiments, analysis of the burst data allows assignment of the identity of incorporated luminescent-labeled nucleotides or that one or more luminescent-labeled nucleotides are distinguished from a plurality of luminescent-labeled nucleotides. would allow The assignment or distinction may depend on any one of emission lifetime, emission intensity, spectral range of emitted photons, or any combination thereof.

FIG. 5 illustrates sequencing of an exemplary template nucleic acid. Sequencing experiments included four luminescently labeled nucleotides: deoxyadenosine (A-AF647) linked to Alexa Fluor® 647, dexoythymidine (T-AF555) linked to Alexa Fluor 555, DyLight® ) with deoxyguanidine linked to 554-R1 (G-D554R1) and dexoycytidine linked to DyLight® 530-R2 (C-D530R2). Nucleotides A-AF647 are excited by excitation energy in the red spectral range, and T, G, and C nucleotides are excited by excitation in the green spectral range. The number of photons detected over a sequencing reaction of approximately 200 seconds is displayed in intensity trace 5-1. Each spike corresponds to a detected burst of luminescence and is marked by a dot. Each burst may correspond to the incorporation of luminescence-labeled nucleotides and contains thousands of detected emissions. Different colored traces can be used to mark different excitation pulses. For example, a purple trace may be used for green excitation pulses and a blue trace may be used for red excitation pulses. The burst of blue trace can be attributed to the incorporation of nucleotide A-AF647 (the only nucleotide with a red emitting molecule in this example).

FIG. 5 shows one way to reduce the raw data to distinguish bursts of the same color (e.g., purple traces between T, G, and C) using the intensity vs. lifetime plot 5-2. is displayed. Each circle represents a burst of purple traces. Each burst was analyzed to determine the luminescent lifetime of the luminescently labeled nucleotide based on the elapsed time between the pulse and the emission of each detected photon. Additionally, each burst was analyzed to determine the emission intensity of the luminescently labeled nucleotide based on the number of photons detected per second. Incorporation events are clustered into three groups corresponding to each of the three luminescently labeled nucleotides. Dark clusters in the lower portion of the plot (plotted area below the dotted line) are assigned to CD530R2 with the longest emission lifetime and lowest emission intensity. Light clusters in the lower portion of the plot (plotted area below the dotted line) are assigned to GD554R1 with intermediate longevity and intensity. Also, the bright clusters in the upper part of the plot (plotted area above the dotted line) are assigned to T-AF555 with the shortest lifetime and highest intensity. FIG. 5 shows an alignment 5-3 of the sequence determined from the data and the known sequence of the template nucleic acid. Vertical lines indicate matches between experimentally determined bases and target sequences. Dashes indicate positions in the template sequence to which no nucleotide was assigned in the determinant sequence, or extra positions in the determinant sequence that do not correspond to any position in the template sequence.

Figure 6 illustrates a second example of sequencing the template nucleic acid. Sequencing experiments were performed using four luminescently labeled nucleotides: deoxyadenosine (A-AF647) linked to Alexa Fluor 647, dexoithymidine (T-AF555) linked to Alexa Fluor 555, Alexa Fluor 647 with deoxyguanidine (G-AF647) linked to , and dexoicytidine (C-AF546) linked to Alexa Fluor®546. Nucleotides A-AF647 and G-AF647 are excited by excitation energy in the red spectral range and T and C nucleotides are excited by excitation in the green spectral range. In this experiment A and G have the same luminescent marker and are not discriminated. In FIG. 6, the number of photons detected over a sequencing reaction of approximately 300 seconds is displayed in intensity trace 6-1. Each spike corresponds to a detected burst of luminescence and is marked by a dot. Each burst may correspond to the incorporation of luminescence-labeled nucleotides and contains thousands of detected emissions. The trace displays the emission detected with the green excitation pulse (corresponding to bases T and C).

FIG. 6 displays one method of reducing the raw data to distinguish T and C using an intensity vs. lifetime plot 6-2. Each circle represents a burst of intensity traces 6-1. Each burst was analyzed to determine the emission lifetime of the luminescently labeled nucleotide based on the elapsed time between the pulse and emission of each detected photon. Additionally, each burst was analyzed to determine the emission intensity of the luminescently labeled nucleotide based on the number of photons detected per second. Incorporation events are clustered into two groups corresponding to each of the two luminescently labeled nucleotides. The dark cluster in the right part of the plot (plotted area to the right of the dotted line) is assigned to C-AF546 with the longest emission lifetime and lowest emission intensity. The bright cluster in the right part of the plot (plotted area to the right of the dotted line) is assigned to T-AF555 with the shortest lifetime and highest intensity. FIG. 6 displays an alignment 6-3 of the sequence determined from the data and the known sequence of the template nucleic acid. Vertical lines indicate matches between experimentally determined bases and target sequences. Horizontal lines indicate positions in the template sequence to which no nucleotides were assigned in the determinant sequence, or extra positions in the determinant sequence that do not correspond to any position in the template sequence.

In some embodiments, one or more of the components of the sequencing reaction are selected from the non-limiting embodiments shown in FIGS. can be prepared and used in sample wells as illustrated in .

Luminescent Labels The terms “luminescent tag,” “luminescent label,” and “luminescent marker” are used interchangeably throughout and relate to molecules that contain one or more luminescent molecules. In some embodiments, the incorporated molecule is, for example, a luminescent molecule that does not have a different luminescent label attached. Typical nucleotides and amino acids are not luminescent or do not emit light within suitable ranges of excitation and emission energies. In some embodiments, the molecule to be incorporated comprises a luminescent label. In certain embodiments, the incorporated molecule is a luminescently labeled nucleotide. In certain embodiments, the molecule to be incorporated is a luminescently labeled amino acid or luminescently labeled tRNA. In some embodiments, a luminescent labeled nucleotide comprises a nucleotide and a luminescent label. In some embodiments, a luminescent labeled nucleotide comprises a nucleotide, a luminescent label, and a linker. In some embodiments, the luminescent label is a fluorophore.

In certain embodiments, the luminescent label and optionally the linker remain attached to the incorporated molecule. In certain embodiments, the luminescent label and optionally the linker are cleaved from the molecule during or after the process of incorporation.

In some embodiments, the luminescent label is a cyanine dye or analogue thereof. In some embodiments, the cyanine dye has the formula:

or a salt, stereoisomer, or tautomer thereof,
wherein A ¹ and A ² together form an optionally substituted aromatic or non-aromatic monocyclic or polycyclic heterocyclic ring;
B ¹ and B ² together form an optionally substituted aromatic or non-aromatic monocyclic or polycyclic heterocyclic ring;
each of R ¹ and R ² is independently hydrogen, optionally substituted alkyl;
Each of L ¹ and L ² is independently hydrogen, optionally substituted alkyl, or L ¹ and L ² taken together are optionally substituted aromatic or non-aromatic form a monocyclic or polycyclic carbocyclic ring.

In some embodiments, the luminescent label is a rhodamine dye or analogue thereof. In some embodiments, the rhodamine dye has the formula:

or a salt, stereoisomer, or tautomer thereof,
wherein each of A ¹ and A ² is independently hydrogen, optionally substituted alkyl, optionally substituted aromatic or non-aromatic heterocyclyl, optionally substituted aromatic is aromatic or non-aromatic carbocyclyl, or optionally substituted carbonyl, or A ¹ and A ² together are optionally substituted aromatic or non-aromatic monocyclic or It forms polycyclic carbocyclic rings.

Each of B ¹ and B ² is independently hydrogen, optionally substituted alkyl, optionally substituted aromatic or non-aromatic heterocyclyl, optionally substituted aromatic or non- aromatic carbocyclyl, or optionally substituted carbonyl, or B ¹ and B ² taken together are an optionally substituted aromatic or non-aromatic monocyclic or polycyclic forming a heterocyclic ring of
each of R ² and R ³ is independently hydrogen, optionally substituted alkyl, optionally substituted aryl, or optionally substituted acyl;
R ⁴ is independently hydrogen, optionally substituted alkyl, optionally substituted aromatic or non-aromatic heterocyclyl, optionally substituted aromatic or non-aromatic carbocyclyl, or is an optionally substituted carbonyl.

In some embodiments, R ⁴ is optionally substituted phenyl. In some embodiments, R ⁴ is optionally substituted phenyl, wherein at least one substituent is optionally substituted carbonyl. In some embodiments, R ⁴ is optionally substituted phenyl, wherein at least one substituent is optionally substituted sulfonyl.

Typically, the luminescent label is an aromatic or heteroaromatic compound, pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, carbazole, thiazole, benzothiazole, phenanthridine, phenoxazine. , porphyrins, quinolines, ethidiums, benzamides, cyanines, carbocyanines, salicylates, anthranilates, coumarins, fluoresceins, rhodamines, or other similar compounds. Exemplary dyes include 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G ), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), xanthene dyes such as fluorescein or rhodamine dyes. Exemplary dyes also include naphthylamine dyes with amino groups in the alpha or beta positions. For example, naphthylamino compounds include 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalenesulfonate, and 2-p-toluidinyl-6-naphthalenesulfonate, 5-(2′-aminoethyl) and aminonaphthalene-1-sulfonic acid (EDANS). Other exemplary dyes include: coumarins such as 3-phenyl-7-isocyanatocoumarin; acridines such as 9-isothiocyanatoacridine and acridine orange; N-(p-(2- benzoxazolyl)phenyl)maleimide; indodicarbocyanine 3 (Cy® 3), (2Z)-2-[(E)-3-[3-(5-carboxypentyl)-1,1- Dimethyl-6,8-disulfobenzo[e]indol-3-ium-2-yl]prop-2-enylidene]-3-ethyl-1,1-dimethyl-8-(trioxidanylsulfanyl)benzo[e]indole -6-sulfonate (Cy® 3.5), 2-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-16,16,18,18- Tetramethyl-6,7,7a,8a,9,10,16,18-octahydrobenzo[2'',3'']indolizino[8'',7'':5',6']pyrano[3 ',2':3,4]pyrido[1,2-a]indol-5-ium-14-sulfonate (Cy® 3B), indodicarbocyanine 5 (Cy® 5), India Cyanine such as dicarbocyanine 5.5 (Cy® 5.5), 3-(-carboxy-pentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CyA); 1H, 5H , 11H,15H-xantheno[2,3,4-ij:5,6,7-i′j′]diquinolidin-18-ium, 9-[2 (or 4)-[[[6-[2,5 -dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4 (or 2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or Texas Red®); BODIPY® dye; benzoxazole; stilbene; and pyrene, and the like.

For nucleotide sequencing, certain combinations of luminescently labeled nucleotides may be preferred. In some embodiments, at least one of the luminescently labeled nucleotides comprises a cyanine dye or analogue thereof. In some embodiments, at least one of the luminescently labeled nucleotides comprises a rhodamine dye or analogue thereof. In some embodiments, at least two of the luminescently labeled nucleotides each comprise a cyanine dye or analogue thereof. In some embodiments, at least two of the luminescently labeled nucleotides each comprise a rhodamine dye or analogue thereof. In some embodiments, at least three of the luminescently labeled nucleotides each comprise a cyanine dye or analogue thereof. In some embodiments, at least three of the luminescently labeled nucleotides each comprise a rhodamine dye or analogue thereof. In some embodiments, at least four of the luminescently labeled nucleotides each comprise a cyanine dye or analogue thereof. In some embodiments, at least four of the luminescently labeled nucleotides each comprise a rhodamine dye or analogue thereof. In some embodiments, three luminescent-labeled nucleotides comprise a cyanine dye or analogue thereof and a fourth luminescent-labeled nucleotide comprises a rhodamine dye or an analogue thereof. In some embodiments, two luminescently labeled nucleotides comprise cyanine dyes or analogues thereof and the third and optionally fourth luminescently labeled nucleotides comprise rhodamine dyes or analogues thereof. In some embodiments, three luminescently labeled nucleotides comprise rhodamine dyes or analogues thereof and the third and optionally fourth luminescently labeled nucleotides comprise cyanine dyes or analogues thereof.

In some embodiments, at least one labeled nucleotide is linked to two or more dyes (eg, two or more copies of the same dye and/or two or more different dyes).

In some embodiments, at least two luminescent-labeled nucleotides absorb the first excitation energy, wherein at least one of the luminescent-labeled nucleotides comprises a cyanine dye or an analogue thereof, and at least one of the luminescent-labeled nucleotides One includes rhodamine dyes or analogues thereof. In some embodiments, at least two luminescent-labeled nucleotides absorb a second excitation energy, wherein at least one of the luminescent-labeled nucleotides comprises a cyanine dye or analogue thereof, and at least one of the luminescent-labeled nucleotides One includes rhodamine dyes or analogues thereof. In some embodiments, at least two luminescent-labeled nucleotides absorb the first excitation energy, wherein at least one of the luminescent-labeled nucleotides comprises a cyanine dye or an analogue thereof, and at least one of the luminescent-labeled nucleotides one comprising a rhodamine dye or analogue thereof, wherein at least two additional luminescently labeled nucleotides absorb the second excitation energy, wherein at least one of the luminescently labeled nucleotides is a cyanine dye or an analogue thereof; and at least one of the luminescently labeled nucleotides comprises a rhodamine dye or analogue thereof.

In some embodiments, at least two of the luminescent-labeled nucleotides absorb the first excitation energy, wherein at least one of the luminescent-labeled nucleotides has an emission lifetime of less than about 1 ns; At least one has an emission lifetime greater than 1 ns. In some embodiments, at least two luminescent-labeled nucleotides absorb a second excitation energy, wherein at least one of the luminescent-labeled nucleotides has an emission lifetime of less than about 1 ns and At least one has an emission lifetime greater than 1 ns. In some embodiments, at least two of the luminescent-labeled nucleotides absorb the first excitation energy, wherein at least one of the luminescent-labeled nucleotides has an emission lifetime of less than about 1 ns; at least one has an emission lifetime greater than 1 ns, and the additional at least two luminescent-labeled nucleotides absorb the second excitation energy, wherein at least one of the luminescent-labeled nucleotides has an emission lifetime of less than about 1 ns have a lifetime, and at least one of the luminescently labeled nucleotides has a luminescent lifetime greater than 1 ns.

In certain embodiments, the luminescent label is a dye selected from Table 1. The dyes listed in Table 1 are non-limiting, and the luminescent labels of the present application may include dyes not listed in Table 1. In certain embodiments, the luminescent label of the one or more luminescently labeled nucleotides is selected from Table 1. In some embodiments, luminescent labels of 4 or more luminescent labeled nucleotides are selected from Table 1.

Dyes can also be classified based on the wavelength of maximum absorption or radiative emission. Exemplary fluorophores are provided in Table 2, grouped in columns according to the approximate wavelength of maximum absorbance. The dyes listed in Table 2 are non-limiting, and the luminescent labels of the present application may include dyes not listed in Table 2. Exact maximum absorption or emission wavelengths may not correspond to the spectral ranges shown. In certain embodiments, the luminescent label of one or more luminescently labeled nucleotides is selected from the "red" group listed in Table 2. In certain embodiments, the luminescent label of one or more luminescently labeled nucleotides is selected from the "green" group listed in Table 2. In certain embodiments, the luminescent label of one or more luminescently labeled nucleotides is selected from the “yellow/orange” group listed in Table 2. In certain embodiments, the luminescent labels of the four nucleotides are all selected from one of the "red", "yellow/orange", or "green" groups listed in Table 2. be. In certain embodiments, the four nucleotide luminescent labels are three selected from the first group of the "red", "yellow/orange", and "green" groups listed in Table 2; Eyes are selected to be selected from the second group of "red", "yellow/orange" and "green" groups listed in Table 2. In certain embodiments, the four nucleotide luminescent labels are two selected from the first group of the "red,""yellow/orange," and "green" groups listed in Table 2, and three An eye and a fourth are selected to be selected from the second group of "red", "yellow/orange" and "green" groups listed in Table 2. In certain embodiments, the four nucleotide luminescent labels are two selected from the first group of the "red,""yellow/orange," and "green" groups listed in Table 2, and three Eyes are selected from a second group of "red", "yellow/orange", and "green" groups listed in Table 2, and a fourth "red" listed in Table 2. , "Yellow/Orange", and "Green" groups.

In certain embodiments, the luminescent label is (dye 101), (dye 102), (dye 103), (dye 104), (dye 105), or (dye 106) of the following formula (NHS ester form), or They may be analogues thereof.

In some embodiments, each sulfonate or carboxylate is independently optionally protonated. In some embodiments, the dyes described above are attached to a linker or nucleotide by formation of an amide bond at the attachment point indicated.

In some embodiments, a luminescent label may comprise first and second chromophores. In some embodiments, the excited state of the first chromophore can be relaxed by energy transfer to the second chromophore. In some embodiments, the energy transfer is Forster Resonance Energy Transfer (FRET). Such FRET pairs can be useful in providing a luminescent label with properties that make it easier to distinguish the label from multiple luminescent labels. In some embodiments, a FRET pair can absorb excitation energy in a first spectral range and emit emission in a second spectral range.

For a set of luminescent-labeled molecules (eg, luminescent-labeled nucleotides), the properties of the luminescent-labeled FRET pair can allow selection of multiple distinguishable molecules (eg, nucleotides). In some embodiments, the second chromophore of the FRET pair has a different emission lifetime than the plurality of other luminescent label molecules. In some embodiments, the second chromophore of the FRET pair has a different emission intensity than the plurality of other luminescent label molecules. In some embodiments, the second chromophore of the FRET pair has a different emission lifetime and emission intensity than the plurality of other luminescent label molecules. In some embodiments, the second chromophore of the FRET pair emits photons in a different spectral range than the plurality of other luminescent label molecules. In some embodiments, the first chromophore of the FRET pair has a different emission lifetime than the plurality of other luminescent label molecules. In some embodiments, a FRET pair can absorb excitation energy in a different spectral range than the plurality of other luminescent label molecules. In some embodiments, a FRET pair can absorb excitation energy in the same spectral range as one or more of the plurality of other luminescent labeled molecules.

In some embodiments, two or more nucleotides may be attached to a luminescent label, where the nucleotides are attached to different positions of the luminescent label. Non-limiting examples can include luminescent molecules containing two independently reactive chemical moieties (e.g., azide, acetylene, carboxyl, amino groups) compatible with the reactive moieties of nucleotide analogues. . In such embodiments, the luminescent label may be attached to the two nucleotide molecules by independent linkers. In some embodiments, a luminescent label may comprise two or more independent connections to two or more nucleotides.

In some embodiments, two or more nucleotides are connected to the luminescent dye by a linker (e.g., a branched linker or linker having two or more reactive sites to which nucleotides and/or dyes can be attached). may be Thus, in some embodiments, two or more nucleotides (eg, of the same type) may be linked to two or more dyes (eg, of the same type).

In some embodiments, luminescent labels may include quantum dots that have luminescent properties. In some embodiments, one or more nucleotides are attached to the quantum dot. In some embodiments, one or more nucleotides are attached to the quantum dot by attachment to different sites on the protein. In some embodiments, the surface of quantum dots is coated with nucleotide molecules. In certain embodiments, the quantum dot is covalently attached to one or more nucleotides (eg, to each component via a reactive moiety). In certain embodiments, a quantum dot is non-covalently attached to one or more nucleotides (eg, to each moiety via a compatible non-covalent binding partner). In some embodiments, the quantum dot surface comprises one or more streptavidin molecules non-covalently bound to one or more biotinylated nucleotides.

In some embodiments, a luminescent label may comprise a protein with luminescent properties. In some embodiments, one or more nucleotides are attached to the photoprotein. In some embodiments, one or more nucleotides are attached to the photoprotein by attachment to different sites on the protein. In certain embodiments, the four nucleotide luminescent labels are such that one nucleotide is labeled with a fluorescent protein and the remaining three nucleotides are labeled with a fluorescent dye (e.g., non-limiting examples in Tables 1 and 2). selected for In certain embodiments, the four nucleotide luminescent labels are such that two nucleotides are labeled with a fluorescent protein and the remaining two nucleotides are labeled with a fluorescent dye (e.g., non-limiting examples in Tables 1 and 2). selected for In certain embodiments, the luminescent labels of the four nucleotides are selected such that three nucleotides are labeled with a fluorescent protein and the remaining nucleotides are labeled with a fluorescent dye (e.g., non-limiting examples in Tables 1 and 2). be done. In some embodiments, the four nucleotide luminescent labels are selected such that all four nucleotides are labeled with a fluorescent protein.

According to some aspects of the present application, luminescent labels (eg, dyes, eg, fluorophores) may damage polymerases of sequencing reactions exposed to excitation light. In some embodiments, this damage occurs during incorporation of luminescent-labeled nucleotides when the luminescent molecule is held in close proximity to the polymerase enzyme. Non-limiting examples of damaging reactions include the formation of covalent bonds between polymerases and luminescent molecules, and the emission of radioactive or non-radioactive decay from luminescent molecules to the enzyme. This may shorten the efficiency of the polymerase and reduce the length over which sequencing is performed.

In some embodiments, the nucleotide and luminescent label are connected by a relatively long linker or linker construct to keep the luminescent label away from the polymerase during incorporation of the labeled nucleotide. The term "linker construct" is used herein to refer to the overall structure connecting the luminescent molecule to the nucleotide and does not include the luminescent molecule or the nucleotide.

In some embodiments, a single linker connects the luminescent molecule to the nucleotide. In some embodiments, a linker has two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) nucleotides attached to each light emitting molecule, or Two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) luminescent molecules are attached to each nucleotide, or two or more (eg, 2, 3 , 4, 5, 6, 7, 8, 9, or 10 or more) nucleotides are It contains one or more branch points to connect to the light emitting molecule.

In some embodiments, the linker configuration determines the distance between the luminescent label and the nucleotide. In some embodiments, the distance is from about 1 nm or 2 nm to about 20 nm. For example, greater than 2 nm, greater than 5 nm, 5-10 nm, greater than 10 nm, 10-15 nm, greater than 15 nm, 15-20 nm, greater than 20 nm. However, the distance between the luminescent label and the nucleotide should not be too long, as the luminescent label must be in the illuminated volume when the nucleotide is retained in the active site of the enzyme. Thus, in some embodiments, the overall linker length is less than 30 nm, less than 25 nm, about 20 nm, or less than 20 nm.

In some embodiments, a protection molecule is included within the linker construct. A protective molecule may be a protein, protein homodimer, protein heterodimer, protein oligomer, polymer, or other molecule capable of protecting the polymerase from damaging reactions that may occur between the enzyme and the luminescent label. Non-limiting examples of protective molecules include proteins (e.g. avidin, streptavidin, traptavidin, NeutrAvidin, ubiquitin), protein complexes (e.g. trypsin: BPTI, barnase: barstar, colicin E9 nuclease: Im9 immunoprotein), Nucleic acids (eg, deoxyribonucleic acids, ribonucleic acids), polysaccharides, lipids, and carbon nanotubes.

In some embodiments, protective molecules are oligonucleotides (eg, DNA oligonucleotides, RNA oligonucleotides, or variants thereof). In some embodiments, the oligonucleotide is single stranded. In some embodiments, the luminescent label is directly or indirectly attached to one end (e.g., the 5' or 3' end) of the single-stranded oligonucleotide, and one or more nucleotides are It is directly or indirectly attached to the other end (eg, the 3' or 5' end) of the single-stranded oligonucleotide. For example, a single-stranded oligonucleotide may include a luminescent label attached to the 5' end of the oligonucleotide and one or more nucleotides attached to the 3' end of the oligonucleotide. In some embodiments, the oligonucleotide is double-stranded (eg, the oligonucleotide comprises two complementary annealed oligonucleotide strands). In some embodiments, the luminescent label is directly or indirectly attached to one end of the double-stranded oligonucleotide and one or more nucleotides are directly or indirectly attached to the other end of the double-stranded nucleotide. Directly or indirectly attached. In some embodiments, the luminescent label is directly or indirectly attached to one strand of the double-stranded oligonucleotide and the one or more nucleotides are directly or indirectly attached to the other strand of the double-stranded nucleotide. Directly or indirectly attached. For example, a double-stranded oligonucleotide may include a luminescent label attached to the 5' end of one strand of the oligonucleotide and one or more nucleotides attached to the 5' end of the other strand.

In some embodiments, the protective molecule is attached to one or more luminescent molecules and to one or more nucleotide molecules. In some embodiments, the luminescent molecule is not contiguous with nucleotides. For example, one or more luminescent molecules may be attached to the first side of the protecting molecule, one or more nucleotides may be attached to the second side of the protecting molecule, and the protecting The first and second sides of the molecule are separated from each other. In some embodiments, they are approximately opposite the protective molecule.

The distance between the point at which the protecting molecule is attached to the luminescent label and the point at which the protecting molecule is attached to the nucleotide may be a linear measurement through space or along the surface of the protecting molecule. It may be a non-linear measurement. The distance between the attachment point of the luminescent label and the nucleotide in the protective molecule can be measured by modeling the three-dimensional structure of the protective molecule. In some embodiments, this distance may be 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 nm or greater. Alternatively, the relative positions of luminescent labels and nucleotides in the protected molecule can be described by treating the structure of the protected molecule as a quadratic surface (eg, ellipsoid, elliptical cylinder). In some embodiments, the luminescent label and the nucleotide are separated by a distance that is at least one-eighth the distance around the oval shape representing the protected molecule. In some embodiments, the luminescent label and the nucleotide are separated by a distance that is at least one-fourth the distance around the oval shape representing the protected molecule. In some embodiments, the luminescent label and the nucleotide are separated by a distance that is at least one-third the distance around the oval shape representing the protective molecule. In some embodiments, the luminescent label and the nucleotide are separated by a distance that is at least one-half the distance around the oval shape representing the protected molecule.

Figure 10 illustrates a non-limiting example 10-1 of a luminescent molecule (200) separated from a nucleotide (250) by a protective molecule (100). The luminescent molecule (200) is connected to the protective molecule (100) via a linker (300), and the linker (300) is attached to the protective molecule (100). Nucleotide (250) is connected to protective molecule (100) via linker (350), and linker (350) is attached to protective molecule (100). A protective molecule may be useful to provide a steric barrier that prevents the luminescent label from gaining access to the polymerase. Protective molecules may be useful to absorb radioactive and non-radioactive decay emitted by the luminescent molecule or to protect the polymerase from such decay. Protective molecules may be useful in providing both a steric and a collapsing barrier between the luminescent label and the polymerase.

The size of the protective molecule should be such that the luminescent label cannot or is difficult to contact directly with the polymerase when the nucleotide is retained within the active site of the enzyme. Also, the size of the protective molecule should be such that when the nucleotide is retained in the active site of the enzyme, the attached luminescent label is present in the irradiated volume to be excited. . The size of the protecting molecule should be selected taking into account the linker chosen to connect the luminescent label to the protecting molecule and the linker chosen to connect the nucleotide to the protecting molecule. The linker used to connect the protective molecule and the luminescent label and the nucleotide (e.g., nucleoside polyphosphate) constitutes a linker construct, the size of which determines when the nucleotide is retained within the active site of the enzyme. , the size should be such that the luminescent label cannot come into direct contact with the polymerase.

The protecting molecule (and/or the linker construct comprising the protecting molecule) is preferably water soluble. In some embodiments, the protecting molecule (and/or the linker construct comprising the protecting molecule) preferably has a net negative charge.

In some embodiments, a label (eg, a luminescent molecule) is not covalently linked to a nucleotide, such that one or more non-covalent linkages attach the label to the nucleotide. In some embodiments, one or more linkers are non-covalently attached to the protecting molecule and/or the light emitting molecule. In some embodiments, one or more linkers are non-covalently attached to the protecting molecules and/or nucleotides. In some embodiments, the luminescent label is covalently attached to the linker, and the linker is non-covalently attached to the protecting molecule (eg, via one or more binding partners). In some embodiments, the nucleotide is covalently attached to the linker, and the linker is non-covalently attached to the protecting molecule (eg, via one or more binding partners).

Figures 10-15 illustrate non-limiting examples of one or more luminescent molecules and one or more nucleotides connected via a protecting molecule, wherein one or more luminescent molecules and One or more nucleotides are non-covalently attached to the protecting molecule.

Figure 10 illustrates a non-limiting example 10-2 comprising elements of 10-1, where the linker non-covalently binds the luminescent molecule to the non-covalent attachment site (110) (dark shape) of the protective molecule. and the second linker comprises a non-covalent ligand (450) that non-covalently attaches the nucleotide to the second non-covalent binding site (120) of the protective molecule. .

FIG. 10 illustrates a non-limiting example 10-3 containing elements of 10-2, where the protective molecule (dark form) contains four independent non-covalent binding sites. A second independent luminescent molecule can be non-covalently attached to the non-covalent binding site (111) of the protective molecule via a non-covalent ligand (401). A second independent luminescent molecule can be non-covalently attached to the non-covalent binding site (121) of the protective molecule via a non-covalent ligand (451). In this embodiment where two independent luminescent molecules and two independent nucleotides are illustrated non-covalently attached at four independent non-covalent binding sites of the protecting molecule, the number of luminescent molecules and nucleotides is was chosen at discretion. In some embodiments, the four independent non-covalent binding sites of the protecting molecule comprise one luminescent molecule at one site and one nucleotide at each of the remaining three sites. In some embodiments, the four independent non-covalent binding sites of the protecting molecule comprise one nucleotide at one site and one luminescent molecule at each of the remaining three sites.

Figure 10 illustrates a non-limiting example 10-4 containing elements of 10-3, where the linker (301) connects a single light-emitting molecule to two independent non-covalent attachment sites of the protecting molecule. containing two independent non-covalent ligands (400, 401) non-covalently attached via (dark shapes) and a linker (351) connecting a single nucleotide to the remaining two independent It contains two independent non-covalent ligands (450, 451) non-covalently attached via non-covalent binding sites.

Figure 11 illustrates a non-limiting example 11-1 containing elements of 10-4, where the linker (301) contains a branch point (302) that allows attachment of two luminescent molecules.
Figure 11 illustrates a non-limiting example 11-2 containing elements of 11-1, where the linker (351) contains a branch point (352) that allows attachment of two nucleotides.

FIG. 11 illustrates a non-limiting example 11-3 containing elements of 11-2, where the linker (302) has two additional branch points (304 ) and the linker (352) contains two additional branch points (354) that allow attachment of four nucleotides.

FIG. 11 illustrates a non-limiting example 11-4 containing elements of 11-3, where each branch point shown at (304) is an additional luminescent molecule for a total of 6 luminescent molecules. Including additional attachment moieties to allow for attachment, each branch point illustrated at (354) includes additional attachment moieties to allow attachment of additional nucleotides for a total of 6 nucleotides.

Figure 12 illustrates a set of non-limiting labeled nucleotide molecules that can be used in sequencing reactions: thymine 12-1, adenine 12-2, cytosine 12-3, and guanine 12-4. In this embodiment, a protective molecule containing four non-covalent binding sites is used to connect six nucleotides of the same type to one or more luminescent molecules. In this embodiment, six nucleotides of the same type are attached to the protective molecule via two separate linkers, each linker being an independent non-nucleotide covalently linked to three nucleotides of the same type. Contains covalent ligands. As illustrated in 12-1, 12-2, and 12-3, a single luminescent molecule is attached to two independent non-covalent bonds non-covalently attached to two independent binding sites of the protective molecule. It is non-covalently attached to the protective molecule via a linker containing a covalent ligand. As illustrated in 12-4, the branch point of the luminescent linker allows for the addition of a second luminescent molecule.

In a sequencing experiment involving four exemplary nucleotides, the luminescent labels at 12-1, 12-2, and 12-3 can comprise three unique luminescent molecules (eg, three different fluorophores). . Since each of these exemplary molecules contains only a single fluorophore, properties used to discriminate thymine, adenine, or cytosine incorporation when no unique fluorophore is used (e.g. , luminescence lifetime, luminescence intensity, emission wavelength) may be highly similar or indistinguishable. Guanine 12-4 is connected to two luminescent molecules. In some embodiments, the luminescent label of 12-4 can comprise two of the same luminescent molecules, the molecule chosen being the fluorophore used in 12-1, 12-2, and 12-3. Different from the fore. In a sequencing reaction containing four unique nucleotides connected to four unique fluorophores, each fluorophore emits one or more unique luminescence that allows each to be distinguished from the others. characteristics (eg, emission lifetime, intensity, emission wavelength, or a combination of two or more thereof). In some embodiments, the two fluorophores of 12-4 can comprise the same two fluorophores and the fluorophores selected are the fluorophores of 12-1, 12-2, and 12-3. is identical to one of In sequencing reactions containing different numbers of unique nucleotides attached to the same luminescent molecule, different numbers of fluorophores have unique properties to allow one nucleotide to be distinguished from the many. (eg, increased emission intensity). Alternatively, the two fluorophores of 12-4 may comprise a FRET pair.

FIG. 12 further illustrates non-limiting examples 12-5 of sequencing experiments and how unique luminescence properties can be used to discriminate from multiple luminescence-labeled nucleotides. ing. The luminescent label attached to each base (thymine, adenine, cytosine, guanine) has luminescent properties (e.g., luminescence lifetime, luminescence intensity, and/or or emission wavelength). Interleaving multiple nucleotides of the same type serves to accelerate the incorporation rate of the sequencing reaction.

Sequencing experiments 12-5 utilizing luminescently labeled nucleotides can be performed in exemplary reaction vessels 12-6. The reaction takes place in a chamber above the waveguide, which acts as a conduit for excitation energy and evanescent waves from the waveguide apply excitation energy to the sample at the bottom of the reaction chamber. The opening not only blocks light diffusing from the waveguide to the bulk sample, and stray light from ambient light and/or the sensor, but also provides a manufacturing pathway for the reaction chamber. The reaction chamber is an etching structure that places the sample at the bottom and within a region of high excitation from the evanescent wave of the waveguide. Selective surface chemistries can be used to provide different compositions to the bottom and sidewalls of the reaction chamber so that the sample can be selectively confined to the bottom of the reaction chamber. is. An exemplary workflow for surface preparation is displayed in FIG. 7, illustrating processes including, among others, processes relating to achieving selective surface chemistry. For example, surface passivation (e.g., as illustrated in the non-limiting embodiment shown in FIG. 8) may be useful during surface functionalization with selective surface substrates (e.g., ) can be provided as illustrated in the non-limiting embodiment shown in FIG.

Specific nucleotide incorporations can be discriminated from the four luminescently labeled nucleotides according to exemplary workflow 12-7 during a sequencing reaction. Over the course of the experiment there are two distinct periods: the pulse period and the detection period. No emitted light is collected during the pulse period lasting 20 picoseconds. After the pulse period, the detection period lasts 10 ns and four time bins capture the radiation events occurring over the detection period (i). The pulse period and detection period constitute one cycle. Emission events are continuously binned and accumulated over the course of one million cycles (ii). The overall distribution of emission events over time bins represents the luminescence lifetime and can be used to match a particular set of data with a known lifetime distribution (iii). In some embodiments, the distribution of radiative events (eg, luminescence lifetimes) does not distinguish one luminescent labeled base from multiple other labeled molecules. In addition to the distribution of radiation events, the quantity of radiation events (eg, emission intensity) can be used to distinguish single molecules from others.

Figure 13 illustrates a non-limiting example 13-1 comprising elements of 10-4, where the non-covalent binding site of the protecting molecule is engineered to be a non-functional binding site (461). there is In some embodiments, the protective molecule for 13-1 may include streptavidin. In some embodiments, the binding site is non-functional in that it cannot non-covalently bind to the biotin molecule. Non-limiting embodiment 13-1 includes a single luminescent molecule attached to the protecting molecule through two independent non-covalent interactions, and a single luminescent molecule attached to the protecting molecule through a single non-covalent interaction. The 6 nucleotides in the sequence are illustrated. In some embodiments, a protective molecule having three functional non-covalent binding sites comprises one or more luminescent molecules attached by a single non-covalent interaction and two independent non-covalent binding It can include one or more nucleotides that are attached by interaction. In some embodiments, a protective molecule having three functional non-covalent binding sites comprises one or more luminescent molecules attached by two independent non-covalent interactions and a single non-covalent bond It can include one or more nucleotides that are attached by interaction.

Figure 13 illustrates a non-limiting example 13-2 containing elements of 10-3, where the two non-covalent binding sites of the protecting molecule are such that they are non-functional binding sites (411, 461). is operated by In some embodiments, the protective molecule of 13-2 may include streptavidin. In some embodiments, the binding site is non-functional in that it cannot non-covalently bind to the biotin molecule. Non-limiting embodiment 13-2 includes a single luminescent molecule attached to the protecting molecule through a single independent non-covalent interaction, and a single luminescent molecule attached to the protecting molecule through a single non-covalent interaction. The 6 nucleotides that do are shown. In some embodiments, a protective molecule having two functional non-covalent binding sites is one or more luminescent molecules attached by a single non-covalent interaction and a single independent non-covalent It can include one or more nucleotides attached by binding interactions. In some embodiments, the two non-functional, non-covalent binding sites are cis or trans binding sites, where "cis binding site" is defined as being in closer proximity than "trans binding site." . Figure 13 further illustrates a non-limiting example 13-3 of generating a streptavidin molecule with two non-functional trans binding sites.

In some embodiments, the linker may be made up of nucleotides. Figure 14 illustrates a non-limiting exemplary reaction scheme in which a luminescent molecule and a nucleotide are connected via a protecting molecule, and the linker that attaches the luminescent molecule and nucleotide to the protecting molecule is a divalent linker. including oligonucleotides (eg, oligonucleotides comprising DNA, RNA, PNA, or modified forms thereof) that are attached via the DNA. A bivalent linker (360) composed of oligonucleotides is non-covalently attached to two independent non-covalent attachment sites of the protective molecule (a). A luminescent molecule (201) fused to an oligonucleotide complementary to the linker (360) is introduced (b) to generate a luminescent labeled protective molecule. A second bivalent linker (361) composed of oligonucleotides is introduced (c). A nucleotide (251) fused to an oligonucleotide complementary to the linker (361) is introduced to generate the final product (d).

Figure 15 illustrates a non-limiting exemplary reaction scheme in which a luminescent molecule is attached to a protective molecule via complementary annealed oligonucleotides, each oligonucleotide strand having a binding site for a protective molecule. contains one non-covalently bound ligand compatible with A first oligonucleotide (362) fused to a luminescent molecule and a non-covalent ligand is annealed to a complementary oligonucleotide (363) fused to the non-covalent ligand (i). Annealing products (364) are bound (ii) to protective molecules and purified. Purified luminescent-labeled protecting molecules are conjugated to nucleotides using any of the techniques or configurations disclosed herein (iii). Also shown in FIG. 15 is an exemplary non-limiting example reaction scheme performed using nucleotides that were labeled with Cy® dye using a double-stranded DNA linker. A sequencing experiment is shown.

In some embodiments, one or more linkers are covalently attached to the protective molecule and/or the luminescent molecule. In some embodiments, one or more linkers are covalently attached to protective molecules and/or nucleotides.

Figures 16-20 illustrate non-limiting examples of luminescent molecules and nucleotides connected via a protecting molecule, where the luminescent molecule and nucleotide are covalently attached to the protecting molecule.

FIG. 16 depicts a reaction scheme 16-1 for generating a generic protection molecule (dark shape), which includes two genetically encoded tags. A genetically encoded tag (325) is designed to form a covalent attachment with a specific reactive group that is fused to a luminescent molecule (i) and another genetically encoded tag (325). The tagged tag (375) is designed to form a covalent attachment with a specific reactive group fused to the nucleotide (ii) to form the final product (c). In FIG. 16, generic substrates containing bioorthogonal functional groups (star shape) such as azides, aldehydes, or alkynes are covalently attached via genetically encoded tags (curves). A non-limiting embodiment of Scheme 16-2 for is further illustrated with non-limiting examples 16-3 of genetically encoded tags and kinetic rate constants.

FIG. 17 illustrates a non-limiting example of Reaction Scheme 17-1 for producing a generic protected molecule (dark shape), where the protected molecule has a reactive group at the N-terminus and a reactive group at the C-terminus. is a protein containing A first reactive group (326) at the first end of the protein is designed to form a covalent attachment with a specific reactive group that is fused to the luminescent molecule. A second reactive group (376) on the remaining end of the protein is designed to form a covalent attachment with a specific reactive group fused to a nucleotide. Non-limiting examples 17-2 of reactive N-terminal groups and reactive C-terminal groups and corresponding reactive groups are further illustrated.

FIG. 18 illustrates a non-limiting example of reaction scheme 18-1 for generating generic protected molecules (dark shapes), which are reactive unnatural amino acids to one portion of the protein. is a protein that contains a reactive unnatural amino acid in another part of the protein. A first reactive unnatural amino acid (327) of the first portion of the protein is designed to form a covalent attachment with a specific reactive group that is fused to the luminescent molecule. A second reactive unnatural amino acid (377) of the second portion of the protein is designed to form a covalent attachment with a specific reactive group fused to the nucleotide. FIG. 18 further illustrates a general non-limiting scheme 18-2 for chemical labeling of target proteins. In this example, an unnatural amino acid bearing a unique biorthogonal functionality (eg, an unnatural amino acid selected from non-limiting Example 18-3) is site-specifically introduced into the protein by genetic code expansion. , which is then chemoselectively labeled with an externally added probe.

Figure 19A is a non-limiting example of a luminescent molecule and a nucleotide separated by a non-protein protecting molecule (101). Non-limiting examples of non-protein protective molecules can include nucleic acid molecules (deoxyribonucleic acid, ribonucleic acid), lipids, and carbon nanotubes. As indicated, the luminescent molecule and the nucleotide are directly attached (eg, covalently attached) to the non-protein protecting molecule. In some embodiments, the luminescent molecule and nucleotide are directly attached to adjacent moieties of the non-protein protecting molecule. For example, in some embodiments, the non-protein protective molecule is a nucleic acid molecule and the luminescent molecule and/or nucleotides are directly attached to the nucleotides of the nucleic acid molecule. In some embodiments, the luminescent molecule and/or nucleotide is attached via a linker to the non-protein protecting molecule that is not a contiguous part of the non-protein protecting molecule. For example, in some embodiments the non-protein protective molecule is a nucleic acid molecule and the luminescent molecule and/or nucleotides are attached to the nucleic acid via a linker.

In some embodiments, luminescent molecules and nucleotides may be attached to non-protein protecting molecules via reactive moieties, as illustrated in FIG. 19B. In this example, the reactive moiety (550) in the luminescent molecule is covalently attached to the non-protein protecting molecule via the corresponding reactive moiety (500) in the non-protein protecting molecule. A reactive moiety (551) at the nucleotide is covalently attached to the non-protein protecting molecule (101) via the corresponding reactive moiety (501) of the non-protein protecting molecule. In some embodiments, reactive moiety 500 and/or reactive moiety 501 are directly attached to the proximal portion of the non-protein protective molecule. In some embodiments, reactive moiety 500 and/or reactive moiety 501 is attached via a linker to a non-protein protecting molecule that is not a contiguous portion of the non-protein protecting molecule.

In some embodiments, protective molecules are composed of protein-protein pairs. A protein-protein pair may constitute any set of polypeptide binding partners (eg, protein-receptor, enzyme-inhibitor, antibody-antigen). Figure 20 illustrates a non-limiting embodiment of protective molecules composed of protein-protein binding pairs. In this non-limiting example, one polypeptide (102) of the binding pair is orthogonally labeled with a luminescent molecule and the second polypeptide (103) is orthogonally linked with nucleotides. Non-limiting examples of protein-protein binding pairs include trypsin-BPTI, barnase-barstar, and colicin E9 nuclease-Im9 immunoprotein.

Figure 21 illustrates non-limiting examples of linker configurations comprising one or more luminescent molecules attached to non-covalently bound ligands. In embodiment 21-1, a first linker layer comprising a non-covalently bound ligand (410), a spacer linker (310), and a reactive moiety (510) comprises a luminescent molecule ( 210). The reactive moieties of the first linker layer can be used to directly attach light emitting molecules or nucleotides via matching reactive moieties. The reactive moieties of the first linker layer can be used to attach the second linker layer via matching reactive moieties. In embodiment 21-2, two light emitting molecules are attached to the first linker layer via a second linker layer, the second linker layer being compatible with the reactive moieties of the first layer. It includes a reactive moiety, a spacer linker (312) containing a branch point, and two reactive moieties (522) that match the reactive moieties of the luminescent molecule. The two reactive moieties of the second linker layer can be used to directly attach light emitting molecules or nucleotide molecules. The two reactive moieties of the second linker layer can be used to attach with the third linker layer to further increase the number of luminescent molecules or nucleotides containing linker configurations. In embodiment 21-3, six luminescent molecules are attached to the second linker layer via a third linker layer attached to each reactive moiety (522), each third linker layer contains a matching reactive moiety (513), a trifunctionalized spacer linker (313), and three reactive moieties (523) that match the reactive moieties of the luminescent molecule.

Thus, the luminescent label may be attached to the molecule directly, eg, by a bond, or via a linker or linker construct. In some embodiments, a linker comprises one or more phosphates. In some embodiments, the nucleotide is connected to the luminescent label by a linker comprising one or more phosphates. As used herein, a linker described as having one or more phosphates is present within the linker structure and is not directly attached to one or more phosphates of a nucleotide Refers to a linker containing one or more phosphates. In some embodiments, the nucleotide is connected to the luminescent label by a linker containing 3 or more phosphates. In some embodiments, the nucleotide is connected to the luminescent label by a linker containing 4 or more phosphates.

In some embodiments, the linker comprises an aliphatic chain. In some embodiments, the linker comprises -(CH ₂ ) _n -, where n is an integer from 1 to 20 inclusive. In some embodiments, n is an integer greater than or equal to 1 and less than or equal to 10. In some embodiments, the linker comprises a heteroaliphatic chain. In some embodiments, the linker comprises a polyethylene glycol moiety. In some embodiments, the linker comprises a polypropylene glycol moiety. In some embodiments, the linker comprises -(CH ₂ CH ₂ O) _n -, where n is an integer from 1 to 20 inclusive. In some embodiments, the linker comprises -(CH ₂ CH ₂ O) _n -, where n is an integer from 1 to 10 inclusive. In some embodiments, the linker comprises -(CH ₂ CH ₂ O) ₄ -. In some embodiments, the linker comprises one or an arylene. In some embodiments, the linker comprises one or more phenylenes (eg, para-substituted phenylenes). In some embodiments, the linker contains a chiral center. In some embodiments, the linker comprises proline or a derivative thereof. In some embodiments, the linker comprises a proline hexamer or derivative thereof. In some embodiments, the linker comprises coumarin or a derivative thereof. In some embodiments, the linker comprises naphthalene or a derivative thereof. In some embodiments, the linker comprises anthracene or a derivative thereof. In some embodiments, the linker comprises polyphenylamide or a derivative thereof. In some embodiments, the linker comprises a chromanone or derivative thereof. In some embodiments, the linker comprises 4-aminopropargyl-L-phenylalanine or a derivative thereof. In some embodiments, a linker comprises a polypeptide.

In some embodiments the linker comprises an oligonucleotide. In some embodiments, the linker comprises two annealed oligonucleotides. In some embodiments, the one or more oligonucleotides comprise deoxyribose, ribose, or locked ribose nucleotides.

In some embodiments, the linker comprises a photostabilizer. In some embodiments, the linker has the formula:

where P ^S is a light stabilizer, the position labeled d is attached to the luminescent label and the position labeled b is attached to the nucleotide. In some embodiments, the position designated d is attached to the luminescent label by a linker described herein. In some embodiments, the position designated b is attached to the nucleotide by a linker as described herein.

In some embodiments, the linker comprises one or more phosphates, aliphatic chains, heteroaliphatic chains, and one or more amides (eg, -C(=O)NH-). In certain embodiments, linkers comprising one or more phosphates and an aliphatic chain can be synthesized via exemplary reaction scheme 22-1 illustrated in FIG. Exemplary linker structures are further illustrated at 22-2 and 22-3. In some embodiments, the linker is selected from those illustrated in Table 3. Certain exemplary linker structures in Table 3 are shown linked to nucleotides (eg, nucleoside hexaphosphates) and/or dyes.

In some embodiments, the linker comprises one or more phosphates, aliphatic chains, heteroaliphatic chains, one or more amides (eg, -C(=O)NH-), and one or more Contains a biotin moiety. In certain embodiments, biotinylated linkers contain one or more functionalizable reactive moieties (eg, acetylene groups, azide groups). In some embodiments, the linker is selected from the non-limiting linkers illustrated in Table 4. Certain exemplary linker structures in Table 4 are shown linked to nucleotides (eg, nucleoside hexaphosphates).

In some embodiments, the linker is synthesized from a precursor comprising a first layer and optionally a second and/or third layer. In some embodiments, the "first layer" contains one or more biotin moieties. In some embodiments, the first layer of linker contains a single biotin moiety. In some embodiments, the first layer of linker contains two biotin moieties. In some embodiments, the first layer contains one or more reactive moieties (eg, azide groups, acetylene groups, carboxyl groups, amino groups). In some embodiments, the first layer of linkers is selected from the structures depicted in Table 5.

In some embodiments, the first layer of linkers can be synthesized according to the following exemplary reaction scheme.

In a further embodiment, the first layer of linkers can be synthesized according to the following exemplary reaction scheme.

In yet further embodiments, the first layer of linkers can be synthesized according to the following exemplary reaction scheme.

In some embodiments, the "second layer" contains one or more reactive moieties (eg, azide groups, acetylene groups, carboxyl groups, amino groups). In some embodiments, the second layer is covalently attached to the first layer of linkers. In some embodiments, the second layer covalently connects the first layer to the third layer. In some embodiments, the third layer contains one or more reactive moieties (eg, azide groups, acetylene groups, carboxyl groups, amino groups). In some embodiments, the third layer is covalently attached to the first layer of linkers. In some embodiments, the third layer is covalently connected to the first layer through the second layer. In some embodiments, the linker comprises first and second layers. In some embodiments, the linker comprises first and third layers. In some embodiments, the linker comprises first, second and third layers. In some embodiments, the second and/or third linker layers are selected from the structures illustrated in Table 6.

In some embodiments, linkers comprising layers beyond the first layer (e.g., second and/or third layers) can be synthesized according to the following exemplary reaction schemes .

In some embodiments, a linker comprising at least second and third layers can be synthesized according to the following exemplary reaction scheme.

Exemplary luminescent labels with biotinylated linkers are displayed in Table 7. It should be understood that different luminescent labels can be used in place of the dyes illustrated in Table 7.

Sample Well (e.g., Nanoaperture) Surface Preparation In certain embodiments, methods of detecting one or more luminescent labeled molecules are performed using molecules confined to a target volume (e.g., reaction volume). do. In some embodiments, the target volume is a region (eg, a nanoaperture) within the sample well. Embodiments of sample wells (eg, nanoapertures) and fabrication of sample wells (eg, nanoapertures) are described elsewhere herein. In some embodiments, a sample well (eg, nanoopening) comprises a bottom surface comprising a first material and sidewalls formed by a plurality of metal or metal oxide layers. In some embodiments, the first material is a transparent material or glass. In some embodiments, the bottom surface is flat. In some embodiments, the bottom surface is a curved well. In some embodiments, the bottom surface includes a portion of the sidewall below sidewalls formed by multiple metal or metal oxide layers. In some embodiments, the first material is fused silica or silicon dioxide. In some embodiments, each of the multiple layers comprises a metal (eg, Al, Ti) or metal oxide (eg, _{Al2O3 , TiO2} , TiN).

Passivation In embodiments where one or more molecules or complexes are immobilized on the bottom surface, it may be desirable to passivate the sidewalls to prevent immobilization to the sidewall surface. In some embodiments, the sidewalls are passivated by depositing a metal or metal oxide barrier layer on the sidewall surface and applying a coating to the barrier layer. In some embodiments, the metal oxide barrier layer comprises aluminum oxide. In some embodiments, depositing includes depositing a metal or metal oxide barrier layer on the sidewall surfaces and the bottom surface. In some embodiments, the depositing step further comprises etching the metal or metal oxide barrier layer from the bottom surface.

In some embodiments, the barrier layer coating comprises phosphonate groups. In some embodiments, the barrier layer coating comprises phosphonate groups with alkyl chains. In some embodiments, the alkyl chain comprises a straight chain saturated hydrocarbon group having 1-20 carbon atoms. In some embodiments, the barrier layer coating comprises hexylphosphonic acid (HPA). In some embodiments, the barrier layer coating comprises polymeric phosphonates. In some embodiments, the barrier layer coating comprises polyvinylphosphonic acid (PVPA). In some embodiments, the barrier layer coating comprises phosphonate groups with substituted alkyl chains. In some embodiments, the alkyl chain includes one or more amides. In some embodiments, the alkyl chains comprise one or more poly(ethylene glycol) chains. In some embodiments, the coating has the formula:

containing a phosphonate group of
wherein n is an integer of 0 or more and 100 or less,
and,

is the point of attachment to the hydrogen or surface. In some embodiments, n is an integer greater than or equal to 3 and less than or equal to 20. In some embodiments, the barrier layer coating comprises a mixture of different types of phosphonate groups. In some embodiments, the barrier layer coating comprises a mixture of phosphonate groups comprising poly(ethylene glycol) chains of different PEG weights.

In some embodiments, the barrier layer comprises nitrodopa groups. In some embodiments, the barrier layer coating has the formula:

containing the group of
wherein R ^N is an optionally substituted alkyl chain, and

is the point of attachment to the hydrogen or surface. In some embodiments, ^RN comprises a polymer. In some embodiments, ^RN comprises poly(lysine) or poly(ethylene glycol). In some embodiments, the barrier layer comprises a copolymer of poly(lysine) comprising lysine monomers, where the lysine monomers independently comprise PEG, nitrodopa groups, phosphonate groups, or primary amines. In some embodiments, the barrier layer comprises a polymer of Formula (P):

In some embodiments, X is -OMe, biotin group, phosphonate or silane. In some embodiments, each of i, j, k and l is independently an integer from 0 to 100 inclusive.

FIG. 8 shows two methods of passivating the metal oxide surface. In 8-1, the metal oxide surface is treated with (2-aminoethyl)phosphonic acid to obtain a surface coated with (2-aminoethyl)phosphonate groups. In a second passivation step, the surface is treated with poly(ethylene glycol) NHS ester. Reaction of the NHS ester with the amine groups of the surface phosphonate groups forms an amide bond to form a surface coated with PEG-functionalized phosphonate groups. In 8-2, a metal oxide surface is treated with PEG-functionalized phosphonic acid to obtain a surface coated with PEG-functionalized phosphonate groups in one step. Exemplary embodiment 8-2 further demonstrates the addition of a second PEG-functionalized phosphonic acid to obtain a surface coated with two types of PEG-functionalized phosphonate groups. In this example, there is a first PEG phosphonic acid with an average molecular weight of 550 and a second PEG phosphonic acid with an average molecular weight of 180. As shown, shorter PEG chain phosphonates can replenish filling into gaps in the surface coverage remaining after treatment with the first PEG phosphonate with longer PEG chains.

Polymerase Immobilization In embodiments in which one or more molecules or complexes are immobilized on the bottom surface, it may be desirable to functionalize the bottom surface to allow binding of one or more molecules or complexes. In some embodiments, the bottom surface comprises clear glass. In some embodiments, the bottom surface comprises fused silica or silicon dioxide. In some embodiments, the bottom surface is functionalized with silane. In some embodiments, the bottom surface is functionalized with an ionically charged polymer. In some embodiments, the ionically charged polymer comprises poly(lysine). In some embodiments, the bottom surface is functionalized with poly(lysine)-graft-poly(ethylene glycol). In some embodiments, the bottom surface is functionalized with biotinylated bovine serum albumin (BSA).

In some embodiments, the bottom surface is functionalized with a coating comprising nitrodopa groups. In some embodiments, the coating has the formula:

containing the group of
wherein R ^N is an optionally substituted alkyl chain, and

is the point of attachment to the hydrogen or surface. In some embodiments, ^RN comprises a polymer. In some embodiments, ^RN comprises poly(lysine) or poly(ethylene glycol). In some embodiments, ^RN comprises biotinylated poly(ethylene glycol). In some embodiments, the coating comprises a copolymer of poly(lysine) comprising lysine monomers that independently comprise PEG, biotinylated PEG, nitrodopa groups, phosphonate groups, or silanes. In some embodiments, the coating comprises a polymer of formula (P):

including.

In some embodiments, X is -OMe, biotin group, phosphonate or silane. In some embodiments, each of i, j, k and l is independently an integer from 0 to 100 inclusive.

In some embodiments, the bottom surface is functionalized with silanes containing alkyl chains. In some embodiments, the bottom surface is functionalized with a silane containing an optionally substituted alkyl chain. In some embodiments, the bottom surface is functionalized with a silane containing poly(ethylene glycol) chains. In some embodiments, the bottom surface is functionalized with a silane containing coupling groups. For example, coupling groups may include chemical moieties such as amine groups, carboxyl groups, hydroxyl groups, sulfhydryl groups, metals, chelating agents, and the like. Alternatively, specific binding members such as biotin, avidin, streptavidin, neutravidin, lectins, SNAP-tags™ or their substrates, associating or binding peptides or proteins, antibodies or antibody fragments, nucleic acids or nucleic acid analogues. may include Additionally or alternatively, the coupling group may optionally include both a chemical functional group and a specific binding member to couple additional groups used to couple or bond with the molecule of interest. may be used for As an example, coupling groups such as biotin may be deposited on the substrate surface and selectively activated in given areas. An intermediate binding agent, such as streptavidin, may then be coupled to the first coupling group. In this particular example, the molecule of interest to be biotinylated is then coupled to streptavidin.

In some embodiments, the bottom surface is functionalized with a silane containing biotin or an analogue thereof. In some embodiments, the bottom surface is functionalized with silanes comprising poly(ethylene glycol) chains, where the poly(ethylene glycol) chains comprise biotin. In some embodiments, the bottom surface is functionalized with a silane mixture in which at least one silane includes biotin and at least one silane does not include biotin. In some embodiments, the mixture is about 10 times less, about 25 times less, about 50 times less, about 100 times less, about 250 times less, about 500 times less, or about 1000 times less than the biotin-free silane. Contains less biotinylated silane.

FIG. 9 shows two exemplary routes to produce a functionalized glass bottom surface. In the top pass, the glass surface is exposed to a mixture of PEG-silane and biotinylated-PEG-silane at a ratio of 250:1. The bottom pass first exposes the glass surface to isocyanato-propyl-triethoxysilane (IPTES). Reaction of isocyanate groups with mixtures of PEG-amine and biotinylated-PEG-amine forms urea linkages, resulting in surfaces with PEG and biotinylated-PEG chains attached to the surface via silane groups.

FIG. 7 shows a non-limiting exemplary process for preparing sample well surfaces from fabricated chips to initiation of sequencing reactions. Sample wells are depicted with bottom (unshaded rectangles) and sidewalls (shaded bars). The sidewalls may consist of multiple layers (eg, Al, Al ₂ O ₃ , Ti, TiO ₂ , TiN). In step (a) the sidewalls are deposited with _a barrier layer of _Al2O3 . The Al ₂ O ₃ barrier layer is then coated with PEG phosphonate groups in step (b), for example by treating the surface with one or more PEG-phosphonic acids. In step (c), the bottom surface is functionalized with, for example, a mixture of PEG-silane and biotinylated-PEG-silane. Ellipses represent individual biotin groups that can provide sites for binding of single molecules or complexes such as polymerase complexes. In step (d) the polymerase complex is attached to the biotin groups on the bottom surface. The polymerase may be bound by a binding agent such as streptavidin and a biotin tag on the polymerase complex. A polymerase complex may further include a template nucleic acid and primers (not shown). Step (e) depicts initiation of the sequencing reaction by exposure of the immobilized polymerase complex to luminescently labeled nucleotides.

A polymerase complex can be immobilized on a surface by exposing the complex to the functionalized surface in a binding mixture. In some embodiments, the binding mixture includes one or more salts. In some embodiments the salt comprises potassium acetate. In some embodiments the salt comprises calcium chloride. In some embodiments, the salt is present at a concentration of about 1 mM to about 10 mM. In some embodiments, the salt is present at a concentration of about 10 mM to about 50 mM. In some embodiments, the salt is present at a concentration of about 50 mM to about 100 mM. In some embodiments, the salt is present at a concentration of about 100 mM to about 250 mM. In some embodiments, the concentration of potassium acetate is about 75 mM. In some embodiments, the concentration of calcium chloride is about 10 mM. In some embodiments, the binding mixture includes a reducing agent. In some embodiments, the reducing agent comprises dithiothreitol (DTT). In some embodiments, the reducing agent is present at a concentration of about 1 mM to about 20 mM. In some embodiments, the dithiothreitol concentration is about 5 mM. In some embodiments, the binding mixture includes a buffer. In some embodiments, the buffer comprises MOPS. In some embodiments, the buffer is present at a concentration of about 10 mM to about 100 mM. In some embodiments, the concentration of MOPS is about 50 mM. In some embodiments, the buffer is present at a pH of about 5.5 to about 6.5. In some embodiments, the buffer is present at a pH of about 6.5 to about 7.5. In some embodiments, the buffer is present at a pH of about 7.5 to about 8.5. In some embodiments, the binding mixture includes deoxynucleotide triphosphates (dNTPs). In some embodiments, deoxynucleotide triphosphates are present at a concentration of 250 nM to 10 μM. In some embodiments, the concentration of dNTPs is about 2 μM. In some embodiments, the binding mixture includes a surfactant. In some embodiments, the detergent is a Tween detergent (eg, Tween 20). In some embodiments, the surfactant is present at a volume percent of about 0.01% to about 0.1%. In some embodiments, the volume percent of Tween is about 0.03%.

Polymerase As used herein, the term "polymerase" generally refers to any enzyme (or polymerizing enzyme) capable of catalyzing a polymerization reaction. Examples of polymerases include, but are not limited to, nucleic acid polymerases, transcriptases or ligases. A polymerase may be a polymerizing enzyme.

Embodiments directed to single-molecule nucleic acid extension (eg, for nucleic acid sequencing) may use any polymerase capable of synthesizing a nucleic acid complementary to a target nucleic acid molecule. In some embodiments, the polymerase may be a DNA polymerase, an RNA polymerase, a reverse transcriptase, and/or one or more mutants or modified forms thereof.

Examples of polymerases include, but are not limited to, DNA polymerases, RNA polymerases, thermostable polymerases, wild-type polymerases, modified polymerases, E. coli DNA polymerase I, T7 DNA polymerase, bacteriophage T4 DNA polymerase ψ29 (psi 29) DNA polymerase, Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, Pwo polymerase, Vent® polymerase, Deep Vent™ polymerase , Ex Taq™ polymerase, LA Taq™ polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tca polymerase, Tih polymerase, Tfi polymerase , Platinum® Taq polymerase, Tbr polymerase, Tfl polymerase, Tth polymerase, Pfuturbo® polymerase, Pyrobest™ polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, 3′→5 'Polymerases with exonuclease activity, and variants, modification products and derivatives thereof. In some embodiments, the polymerase is a single subunit polymerase. Non-limiting examples of DNA polymerases and their properties are found, inter alia, in DNA Replication 2nd Edition, Kornberg and Baker, W.; H. Freeman, New York, N.W. Y. (1991).

Upon base pairing between the nucleobases of the target nucleic acid and the complementary dNTP, the polymerase creates a phosphodiester bond between the 3' hydroxyl terminus of the newly synthesized strand and the alpha phosphate of the dNTP. Formation incorporates dNTPs into newly synthesized nucleic acid strands. In instances where the luminescent tag conjugated to the dNTP is a fluorophore, its presence is signaled by excitation and a pulse of emission detected during or after the incorporation step. For detection labels conjugated to terminal (gamma) phosphates of dNTPs, incorporation of dNTPs into newly synthesized strands releases β and γ phosphates as well as detection labels that diffuse freely in the sample well. thus reducing the detected emission from the fluorophore.

In some embodiments, the polymerase is a highly processable polymerase. However, in some embodiments, the polymerase is a polymerase with reduced processability. Polymerase processability generally refers to the ability of a polymerase to sequentially incorporate dNTPs into a nucleic acid template without releasing the nucleic acid template.

In some embodiments, the polymerase is a polymerase with low 5'-3' exonuclease activity and/or 3'-5' exonuclease. In some embodiments, the polymerase is modified (eg, by amino acid substitutions) to have reduced 5'-3' exonuclease activity and/or 3'-5' activity compared to the corresponding wild-type polymerase. be. Further non-limiting examples of DNA polymerases include 9°Nm™ DNA polymerase (New England Biolabs) and the P680G mutant of Klenow exopolymerase (Tuske et al. (2000) JBC 275(31):23759). ~23768). In some embodiments, polymerases with reduced processability increase the accuracy of sequencing templates that contain one or more stretches of nucleotide repeats (eg, two or more consecutive bases of the same type).

In some embodiments, the polymerase is a polymerase that has a higher affinity for labeled nucleotides than for unlabeled nucleic acids.
Embodiments directed to unimolecular RNA extension (eg, for RNA sequencing) may use any reverse transcriptase capable of synthesizing complementary DNA (cDNA) from an RNA template. In such embodiments, a reverse transcriptase may function similarly to a polymerase in that it can synthesize cDNA from an RNA template via incorporation of dNTPs into a reverse transcription primer annealed to the RNA template. . The cDNA can then be subjected to a sequencing reaction, the sequence of which is determined either above and elsewhere herein. The determined sequence of the cDNA may then be used through sequence complementarity to determine the sequence of the original RNA template. Examples of reverse transcriptases include Moloney murine leukemia virus reverse transcriptase (M-MLV), avian myeloblastosis virus (AMV) reverse transcriptase, human immunodeficiency virus reverse transcriptase (HIV-1) and telomerase reverse transcriptase. enzymes.

The processability, exonuclease activity, relative affinity for different types of nucleic acids, or other properties of nucleic acid polymerases may be increased by those skilled in the art by mutations or other modifications relative to the corresponding wild-type polymerases. , or may be reduced.

Templates The present disclosure provides devices, systems and methods for detecting biomolecules, such as nucleic acid molecules, or subunits thereof. Such detection may include sequencing. Biomolecules may be extracted from a biological sample obtained from a subject (eg, a human or other subject). In some embodiments, the subject may be a patient. In some embodiments, a target nucleic acid can be detected and/or sequenced for diagnostic, prognostic and/or therapeutic purposes. In some embodiments, information regarding sequencing assays may be useful to aid in the diagnosis, prognosis, and/or treatment of diseases or conditions. In some embodiments, a subject may be suspected of having a medical condition, such as a disease (eg, cancer). In some embodiments, the subject may be undergoing treatment for a disease.

In some embodiments, a biological sample can be extracted from a subject's bodily fluids or tissues, such as breath, saliva, urine, blood (e.g., whole blood or plasma), stool, or other bodily fluids or biopsy samples. . In some examples, one or more nucleic acid molecules are extracted from a subject's bodily fluid or tissue. The one or more nucleic acids may be extracted from one or more cells obtained from the subject, such as a portion of the subject's tissue, or may be obtained from the subject's acellular body fluid, such as whole blood. good.

A biological sample may be processed in preparation for detection (eg, sequencing). Such processing can include isolation and/or purification of biomolecules (eg, nucleic acid molecules) from a biological sample and generation of more copies of biomolecules. In some examples, one or more nucleic acid molecules are isolated and purified from a subject's bodily fluids or tissues and amplified by nucleic acid amplification such as the polymerase chain reaction (PCR). One or more nucleic acid molecules or subunits thereof may then be identified, such as through sequencing. However, in some embodiments, nucleic acid samples may be evaluated (eg, sequenced) as described in this application without the need for amplification.

As described in this application, sequencing involves the determination of individual subunits of a template biomolecule (e.g., a nucleic acid molecule) to identify another biomolecule complementary or similar to the template. Determination can involve synthesis, eg, by synthesizing a nucleic acid molecule that is complementary to the template nucleic acid molecule and identifying nucleotide incorporations over time (eg, sequencing by synthesis). Alternatively, sequencing may involve direct identification of individual subunits of biomolecules.

During sequencing, signals indicative of individual subunits of a biomolecule may be collected in memory and processed in real-time or at a later time to determine the sequence of the biomolecule. Such processing may involve comparison of the signal to a reference signal allowing identification of individual subunits, optionally resulting in a readout. The reads are of sufficient length (e.g., at least about 30, 50, 100 base pairs (e.g., at least about 30, 50, 100 base pairs ( bp) or more).

Sequence reads may be used to reconstruct (eg, by alignment) longer regions of the genome of interest. Reads may be used to reconstruct chromosomal regions, entire chromosomes, or entire genomes. Sequence reads or larger sequences generated from such reads may be used to analyze the subject's genome, such as to identify variants or polymorphisms. Examples of variants include, but are not limited to, single nucleotide polymorphisms (SNPs), including tandem SNPs, small multiple nucleotide deletions or insertions (indels or deletion insertion polymorphisms (DIP) multinucleotide polymorphisms (MNPs), short tandem repeats (STRs), deletions including microdeletions, insertions, e.g. microinsertions, structural alterations e.g. duplications, inversions, translocations , multiplication, compound multiple site variants, copy number variation (CNV). A genomic sequence may contain a combination of variants. For example, a genomic sequence can include a combination of one or more SNPs and one or more CNVs.

The term "genome" generally refers to the entire genetic information of an organism. The genome may be either DNA encoded or RNA encoded. A genome may include coding regions that encode proteins and non-coding regions. A genome may include the sequences of all chromosomes together in an organism. For example, the human genome has a total of 46 chromosomes. All these sequences together make up the human genome. In some embodiments, the entire genome is sequenced. However, in some embodiments, sequence information of a subset of the genome (e.g., one or several chromosomes or regions thereof) or one or less genes (or fragments thereof) may be used for diagnostic applications, prognostic applications and/or sufficient for therapeutic application.

Nucleic acid sequencing of multiple single-stranded target nucleic acid templates, where multiple sample wells (e.g., nano-apertures) are available, such as in devices described elsewhere herein can be completed in Each sample well may be provided with a single-stranded target nucleic acid template and a sequencing reaction may be completed within each sample well. Appropriate reagents required for nucleic acid synthesis (e.g., dNTPs, sequencing primers, polymerases, cofactors, appropriate buffers, etc.) may be contacted with each sample well during the primer extension reaction, and the sequence Determining reactions may proceed in each sample well. In some embodiments, multiple sample wells are contacted with all appropriate dNTPs simultaneously. In other embodiments, multiple sample wells are separately contacted with the appropriate dNTP and washed between contact with each different dNTP. Incorporated dNTPs are detectable in each sample well, and sequences determined for single-stranded target nucleic acids in each sample well are detected, as described elsewhere herein. can be

Although some embodiments can be directed to diagnostic testing by detecting single molecules in a specimen, the single molecule detection capabilities of the present disclosure can be used to detect, for example, one or more nucleic acid segments of a gene. It is also understood by the inventors that polypeptide (eg protein) sequencing or nucleic acid (eg DNA, RNA) sequencing may be performed.

In some aspects, the methods described herein may be performed using one or more devices or apparatuses described in more detail below.
Instrument Overview We believe that a compact, high-speed instrument for single molecule or particle detection and quantification will reduce the cost of performing complex quantitative measurements of biological and/or chemical samples, and reduce biochemical We further recognize and understand that the speed of technological discovery can be rapidly increased. Moreover, an easily transportable, cost-effective device could not only change the way bioassays are performed in developed countries, but also a basic diagnostic test that could dramatically improve the health and well-being of people in developing regions. for the first time. For example, the embodiments described herein can be used for blood, urine and/or saliva diagnostic tests that can be used by individuals at home or by physicians in remote clinics in developing countries. .

Pixelated sensor devices with a large number of pixels (eg, hundreds, thousands, tens of thousands or more) allow parallel detection of multiple individual molecules or particles. Molecules may be, by way of example and not limitation, proteins and/or DNA. Furthermore, high speed devices capable of acquiring data at over 100 frames per second enable the detection and analysis of dynamic processes or changes that occur over time within the sample under analysis.

The inventors have recognized and understood that one hurdle that has prevented bioassay devices from becoming more compact has been the need to filter excitation light that causes unwanted detection events at the sensor. there is Optical filters used to transmit the desired signal light (emission) and sufficiently block the excitation light are thick, bulky, expensive, cannot withstand variations in the angle of incidence of the light, and are a hindrance to miniaturization. may become. However, the inventors recognize that the use of a pulsed excitation source may reduce the need for such filtering, or in some cases eliminate the need for such filtering entirely; got it. By using a sensor that can determine the time at which a photon is detected for an excitation light pulse, the signal light is separated from the excitation light based on the time the photon is received rather than the spectrum of the received light. be able to. Thus, the need for bulky optical filters is reduced and/or eliminated in some embodiments.

The inventors recognize and understand that luminescence lifetime measurements can also be used to identify molecules present in a sample. An optical sensor capable of detecting when a photon is detected can use statistics gathered from many events to measure the luminescence lifetime of a molecule being excited by the excitation light. In some embodiments, luminescence lifetime measurements may be made in addition to spectral measurements of luminescence. Alternatively, spectral measurement of emission may be omitted entirely in the step of identifying sample molecules. Luminescence lifetime measurements may be made using a pulsed excitation source. Additionally, luminescence lifetime measurements may be made using an integrated device that includes the sensor or a device in a system where the light source is separate from the integrated device.

The inventors also found that the integration of sample wells (e.g., nano-apertures) and sensors into a single integrated device capable of measuring luminescent light emitted from a biological sample is a disposable biochemical analysis chip. It is also recognized and understood to reduce the cost of fabricating such a device so that a can be formed. A disposable, single-use integrated device that interfaces with a base instrument can be used anywhere in the world without the constraints of requiring high-cost biological laboratories for sample analysis. Thus, automated biochemical analysis can be brought to areas of the world where quantitative analysis of biological samples was previously not possible. For example, a blood test for infants places a blood sample into a disposable all-in-one device, places the disposable all-in-one device for analysis into a small portable base instrument, and processes the results by computer for immediate review by the user. can be done by The data can also be transmitted over a data network to a remote location for analysis and/or archived for subsequent clinical analysis.

The inventors also recognize and understand that disposable, single-use devices can be made easier and less expensive by not including a light source on the chip. Alternatively, the light source may be a reusable component integrated into a sample analysis system in conjunction with a disposable chip.

The inventors have also found that when a sample is tagged with multiple different types of luminescent markers, any suitable property of the luminescent markers can be used to identify the type of marker present within a particular pixel of the chip. I also recognize and understand that it can be used. For example, properties of the luminescence emitted by the marker and/or properties of the excitation absorption may be used to identify the marker. In some embodiments, the radiant energy of the emitted light (which is directly related to the wavelength of the light) may be used to distinguish between the first type of marker and the second type of marker. Additionally or alternatively, luminescence lifetime measurements can be used to identify the type of marker present at a particular pixel. In some embodiments, luminescence lifetime measurements may be made with a pulsed excitation source using a sensor capable of distinguishing times when photons are detected with sufficient resolution to obtain lifetime information. Additionally or alternatively, the energy of the excitation light absorbed by different types of markers may be used to identify the type of marker present at a particular pixel. For example, a first marker can absorb light of a first wavelength but not likewise absorb light of a second wavelength, while a second marker can absorb light of a second wavelength. It can absorb light, but it cannot absorb light of the first wavelength as well. Thus, if more than one excitation light source, each with a different excitation energy, can be used to illuminate the sample in an interleaved fashion, the absorbed energy of the markers will determine which types of markers are present in the sample. can be used to identify Different markers can also have different emission intensities. Therefore, the detected intensity of luminescence can also be used to identify the type of marker present at a particular pixel.

I. System Overview A system includes an integrated device and equipment configured to interface with the integrated device. The integrated device includes an array of pixels, each pixel including a sample well and at least one sensor. A surface of the integrated device includes a plurality of sample wells, the sample wells configured to receive samples from specimens placed on the surface of the integrated device. A specimen may contain multiple samples, and in some embodiments may contain different types of samples. The multiple sample wells can have a suitable size and shape such that at least some sample wells receive one sample from the specimen. In some embodiments, the number of samples in the sample wells is distributed among the sample wells such that some sample wells contain one sample and others contain zero, two or more samples. may be

In some embodiments, the specimen may contain a plurality of single-stranded DNA templates, and individual sample wells on the surface of the integrated device are sized and shaped to receive the single-stranded DNA templates. good too. The single-stranded DNA template may be distributed among the sample wells of the integrated device such that at least some sample wells of the integrated device contain the single-stranded DNA template. The specimen can also contain tagged dNTPs, which then enter the sample well and allow identification of the nucleotides as they are incorporated into a strand of DNA complementary to the single-stranded DNA template in the sample well. can be made possible. "Sample" in such instances can refer to both single-stranded DNA and tagged dNTPs currently being incorporated by the polymerase. In some embodiments, the specimen may contain a single-stranded DNA template, and tagged dNTPs may then be introduced into the sample well as nucleotides are incorporated into the complementary strand of DNA within the sample well. . In this way, the timing of nucleotide incorporation can be controlled by when tagged dNTPs are introduced into the sample wells of the integrated device.

Excitation energy is provided from an excitation source located separately from the pixel array of the integrated device. Excitation energy is directed, at least in part, by elements of the integrated device into one or more pixels to illuminate an illumination region within the sample well. The markers or tags can then emit radiant energy when within the illuminated area and in response to illumination by the excitation energy. In some embodiments, the one or more excitation sources are part of an instrument of a system in which the instrument components and integrated devices are configured to direct excitation energy to one or more pixels. .

The radiant energy emitted by the sample can then be detected by one or more sensors within one pixel of the integrated device. A characteristic of the detected radiant energy can provide an indication for identifying markers associated with the radiant energy. Such properties include any suitable type of property, including the time of arrival of photons detected by the sensor, the amount of photons accumulated over time by the sensor, and/or the distribution of photons across two or more sensors. obtain. In some embodiments, the sensor may have a configuration that allows detection of one or more timing properties associated with the sample's radiant energy (eg, fluorescence lifetime). The sensor can detect the distribution of photon arrival times after a pulse of excitation energy has propagated through the integrated device, and the distribution of arrival times can provide an indication of the timing characteristics of the sample's radiant energy. (eg surrogate for fluorescence lifetime). In some embodiments, one or more sensors provide an indication of the probability of radiant energy emitted by a marker or tag (eg, fluorescence intensity). In some embodiments, the multiple sensors may be sized and arranged to capture the spatial distribution of radiant energy. Output signals from one or more sensors can then be used to distinguish one marker from among multiple markers, and multiple markers can be used to distinguish samples within a specimen. In some embodiments, the sample may be excited with multiple excitation energies, and the radiant energy emitted by the sample in response to the multiple excitation energies and/or the timing characteristics of the radiant energy can distinguish one marker from multiple markers.

A schematic diagram of system 23-100 is illustrated in FIGS. 23A and 23B. The system includes both an integrated device 23-102 that interfaces with equipment 23-104. In some embodiments, instrument 23-104 may include one or more excitation sources 23-106 integrated as part of instrument 23-104. In some embodiments, the excitation source may be external to both the equipment 23-104 and the integrated device 23-102, with the equipment 23-104 receiving excitation energy from the excitation source and integrating it It may be configured to point to a device. The integrated device can interface with the instrument using any suitable socket for receiving the integrated device and keeping it in precise optical alignment with the excitation source. Excitation source 23-106 may be configured to provide excitation energy to integrated device 23-102. As schematically illustrated in FIG. 23B, integrated device 23-102 has a plurality of pixels, and at least some of pixels 23-112 are capable of independent analysis of the sample. Such pixels 23-112 are referred to as "passive source pixels" because they receive excitation energy from a source 23-106 that is separate from the pixel, which excites multiple pixels. can be Pixels 23-112 are configured to receive a sample and a sensor 23-110 for detecting radiant energy emitted by the sample in response to illumination of the sample with excitation energy provided by excitation source 23-106. sample wells 23-108. The sample well 23-108 can hold the sample near the surface of the integrated device 23-102 to facilitate application of excitation energy to the sample and detection of radiant energy from the sample.

Optical elements for directing and coupling excitation energy to the sample well 23-108 are present in both the integrated device 23-102 and instrument 23-104. Such source-to-well elements include one or more grating couplers on the integrated device 23-102, and equipment 23 for coupling excitation energy to the integrated device. Waveguides may be provided for applying excitation energy from -104 to the sample wells of pixels 23-112. In some embodiments, elements on the integrated device may act to direct radiant energy from the sample well to the sensor. The sample well 23-108 is part of the excitation source-to-well optics and the sample well-to-sensor optics are on the integrated device 23-102. be. The excitation source 23-106 and some of the source-to-well components reside in the instrument 23-104. In some embodiments, a single component may play a role in both coupling excitation energy to sample well 23-108 and applying radiant energy from sample well 23-108 to sensor 23-110. good. Examples of suitable components for inclusion in an integrated device for coupling excitation energy to the sample well and/or directing radiant energy to the sensor include the "Integrated Device for Probing, Detecting and Analyzing Molecules". No. 14/821,688, entitled INTEGRATED DEVICE FOR PROBING, DETECTING AND ANALYZING MOLECULES, and "Integrated Device with External Light Source for Probing, Detecting, and Analyzing Molecules." No. 14/543,865 entitled INTEGRATED DEVICE WITH EXTERNAL LIGHT SOURCE FOR PROBING, DETECTING, AND ANALYZING MOLECULES, both of which are incorporated by reference in their entirety. be done.

As illustrated in FIG. 23B, the integrated device comprises a plurality of pixels, each pixel 23-112 being associated with its own individual sample well 23-108 and at least one sensor 23-110. A plurality of pixels may be arranged in an array, and there may be any suitable number of pixels in the array. The number of pixels in the integrated device 23-102 may range from approximately 10,000 pixels to 1,000,000 pixels, or any value or range of values within this range. In some embodiments, the pixels may be arranged in a 512 pixel by 512 pixel array. Integrated device 23-102 and instrument 23-104 may include multi-channel high-speed communication links for handling data associated with large pixel arrays (eg, greater than 10,000 pixels).

Appliance 23-104 interfaces with integrated device 23-102 through integrated device interface 23-114. The integrated device interface 23-114 positions the integrated device 23-102 relative to the instrument 23-104 to improve coupling of excitation energy from the excitation source 23-106 to the integrated device 23-102. and/or components for alignment. Excitation sources 23-106 may be any suitable light source arranged to apply excitation energy to at least one sample well. Examples of suitable excitation sources include the INTEGRATED DEVICE FOR MOLECULAR PROBING, DETECTION AND ANALYSIS, which is incorporated by reference in its entirety.
14/821,688 entitled PROBING, DETECTING AND ANALYZING MOLECULES. In some embodiments, excitation source 23-106 includes multiple excitation sources combined to apply excitation energy to integrated device 23-102. Multiple excitation sources may be configured to generate multiple excitation energies or wavelengths. The integrated device interface 23-114 can receive readout signals from sensors in pixels on the integrated device. The integrated device interface 23-114 may be designed so that the integrated device attaches to equipment by securing the integrated device to the integrated device interface 23-114.

Device 23-104 includes a user interface 23-116 for controlling operation of device 23-104. User interfaces 23-116 are configured to allow a user to enter information into the device, such as commands and/or settings used to control device functions. In some embodiments, user interface 23-116 may include buttons, switches, dials, and a microphone for voice commands. Additionally, the user interface 23-116 allows the user to receive feedback regarding the performance of the instrument and/or the integrated device, such as information obtained by proper alignment and/or readout signals from sensors on the integrated device. can be made possible. In some embodiments, user interfaces 23-116 may provide feedback using speakers to provide audible feedback and indicator lights and/or display screens to provide visual feedback. In some embodiments, the equipment 23-104 includes a computer interface 23-118 used to connect with the computing device 23-120. Any suitable computer interface 23-118 and computing device 23-120 may be used. For example, computer interface 23-118 may be a USB interface or a Firewire interface. Computing devices 23-120 may be any general purpose computer such as a laptop or desktop computer. Computer interface 23-118 facilitates communication of information between appliance 23-104 and computing device 23-120. Input information for controlling and/or configuring the instrument 23-104 may be provided through a computing device 23-120 connected to the instrument's computer interface 23-118. Output information may be received by computing device 23-120 through computer interface 23-118. Such output information may include feedback on the performance of instruments 23-104 and/or integrated devices 23-112, as well as information from readout signals of sensors 23-110. Instruments 23-104 may also include processing devices 23-122 for analyzing data received from sensors 23-110 and/or transmitting control signals to excitation sources 23-106. In some embodiments, the processing devices 23-122 are general purpose processors, specially adapted processors (e.g., central processing units (CPUs) such as one or more microprocessor or microcontroller cores, field programmable gate arrays (FPGA)). , an application-specific integrated circuit (ASIC), a custom integrated circuit, a digital signal processor (DSP), or combinations thereof). In some embodiments, processing of data from sensors 23-110 may be performed by both processing device 23-122 and external computing device 23-120. In other embodiments, computing device 23-120 may be omitted and processing of data from sensor 23-110 may be performed entirely by processing device 23-122.

A cross-sectional schematic view of the integrated device 24-102 illustrating columns of pixels is shown in FIG. 24A. Each pixel 24-112 includes a sample well 24-108 and a sensor 24-110. The sensor 24-110 may be aligned and positioned with respect to the sample well 24-112 such that the sensor 24-110 receives radiant energy emitted by the sample within the sample well 24-112. Examples of suitable sensors can be found in U.S. patent application Ser. 821,656.

An excitation source coupled to the integrated device can provide excitation energy to one or more pixels of the integrated device 24-102. FIG. 24B is a schematic illustrating coupling of an excitation source 24-106 to the integrated device 24-102 to provide excitation energy 24-130 (shown in dashed line) to the integrated device 24-102. It is a diagram. FIG. 24B illustrates the path of excitation energy from excitation energy source 24-106 to sample well 24-108 in pixel 24-112. A component located remotely from the integrated device can be used to position and align the excitation sources 24-106 with respect to the integrated device. Such components can include optical components including lenses, mirrors, prisms, apertures, attenuators, and/or optical fibers. Additional mechanical components may be included in the instrument to allow control of one or more alignment components. Such mechanical components may include actuators, stepper motors, and/or knobs.

The integrated device includes components that direct excitation energy 24-130 to pixels in the integrated device. Within each pixel 24-112 excitation energy is coupled to the sample well 24-108 associated with the pixel. Although FIG. 24B illustrates excitation energy coupling to each sample well in a row of pixels, in some embodiments excitation energy may not be coupled to all of the pixels in a row. good. In some embodiments, excitation energy may be coupled to sample wells in a portion of pixels or a row of pixels of an integrated device. Excitation energy can illuminate a sample located within the sample well. A sample can reach an excited state in response to irradiation with excitation energy. When the sample is in an excited state, the sample can emit radiant energy, which can be detected by the sensor. FIG. 24B schematically illustrates the path (shown as a solid line) of radiant energy 24-140 from sample well 24-108 of pixel 24-112 to sensor 24-110. Sensors 24-110 in pixels 24-112 may be constructed and arranged to detect radiant energy from sample wells 24-108. In some embodiments, sensor 24-110 may include multiple sub-sensors.

A sample to be analyzed can be introduced into sample well 24-108 of pixel 24-112. The sample may be a biological sample or any other suitable sample such as a chemical sample. A sample may contain multiple molecules, and a sample well may be configured to isolate a single molecule. In some examples, the dimensions of the sample well can serve to confine a single molecule within the sample well, allowing measurements to be made on a single molecule. The excitation source 24-106 is for exciting the sample or at least one luminescent marker attached to or otherwise associated with the sample while the sample is within the illuminated area in the sample well 24-108. Additionally, it may be configured to apply excitation energy to the sample well 24-108.

At least one sample in the well may emit light when the excitation source applies excitation energy to the sample well, and the resulting emission may be detected by the sensor. As used herein, the phrases "the sample can emit light" or "the sample can emit radiation" or "emission from the sample" refer to a luminescent tag, marker, or reporter, the sample itself, or the sample. It means that the associated reaction product can produce radiation.

One or more components of the integrated device can direct radiant energy to the sensor. The radiant energy can be detected by the sensor and converted into at least one electrical signal. Electrical signals may be transmitted along conductive lines in circuits of an integrated device connected to an instrument through an integrated device interface, such as integrated device interface 23-114 of instrument 23-104 shown in FIG. 23B. The electrical signal can then be processed and/or analyzed. Processing or analysis of the electrical signals may occur on the instrument 23-104 or in a suitable computing device remote from the instrument, such as the computing device 23-120 shown in FIG. 23B.

Parallel analysis of the sample in the sample well is performed during operation by exciting the sample in the well with an excitation source and detecting a signal from the sample emission with the sensor. Radiant energy from the sample can be detected by a corresponding sensor and converted into at least one electrical signal. The resulting signal may be processed on an integrated device in some embodiments or transmitted to an instrument for processing by a processing device and/or computing device. Signals from sample wells can be received and processed independently of signals associated with other pixels.

In some embodiments, the sample may be labeled with one or more markers, and the emissions associated with the markers are distinguishable by the instrument. For example, the sensor may be configured to convert photons from the radiant energy into electrons to form an electrical signal that can be used to determine the lifetime dependent radiant energy from a particular marker. By labeling samples using markers with different lifetimes, specific samples can be identified based on the resulting electrical signal detected by the sensor.

A sample may contain multiple types of molecules, and different luminescent markers can be uniquely associated with a molecule type. During or after excitation, a luminescent marker can emit radiant energy. One or more characteristics of the radiant energy can be used to identify one or more types of molecules in the sample. Radiant energy attributes used to distinguish between types of molecules may include fluorescence lifetime values, intensities, and/or emission wavelengths. A sensor can detect photons, including photons of radiant energy, and provide an electrical signal indicative of one or more of these characteristics. In some embodiments, the electrical signal from the sensor can provide information about the distribution of photon arrival times over one or more time intervals. The distribution of photon arrival times may correspond to when photons are detected after a pulse of excitation energy is emitted by the excitation source. The time interval value may correspond to the number of photons detected during the time interval. Relative values over multiple time intervals can provide an indication of the time characteristics (eg, lifetime) of the radiant energy. Analysis of the sample may include distinguishing between markers by comparing values at two or more different time intervals within the distribution. In some embodiments, an indication of intensity can be provided by determining the number of photons across all time bins in the distribution.

II. Integrated Device An integrated device may be configured to receive excitation energy from an external excitation energy source. In some embodiments, regions of the device may be used to couple to excitation energy sources located remotely from the integrated device. A component of the integrated device can direct excitation energy from the excitation source coupling region to at least one pixel. In some embodiments, at least one waveguide may be configured to apply excitation energy to at least one pixel having a sample well. A sample within the sample well can emit radiant energy in response to irradiation by the excitation energy. One or more sensors within the pixels are configured to receive the radiant energy.

Components and/or layers of integrated device 25-200 according to some embodiments shown in FIG. including. A sample well 25-203 may be formed in the sample well layer 25-201 of the integrated device 25-200. In some embodiments, sample well layers 25-201 may be metal. The sample wells 25-203 may have a dimension D _tv that may indicate the cross-sectional dimension of the sample well. The sample wells 25-203 may act as nano-apertures and may have one or more sub-wavelength dimensions that provide a field enhancement effect that enhances the excitation intensity of the sample in the sample wells 25-203. Waveguide 25-220 is configured to apply excitation energy to sample well 25-203 from an excitation source 25-230 located remotely from device 25-200. A waveguide 25-220 may be formed in a layer between the sample well layer 25-201 and the sensor 25-275. The design of integrated device 25-200 allows sensor 25-275 to collect emitted light from the sample in sample well 25-203. At least sometimes, the sample absorbs the excitation energy and emits photons having less energy than the excitation energy, called radiant energy or luminescence.

Having sample well 25-203 and sensor 25-275 in integrated device 25-200 can reduce the optical distance that light travels from sample well 25-203 to sensor 25-215. The dimensions of the integrated device 25-200 or components within the device may be configured for a particular optical distance. Optical properties of the components of the device and/or the material of one or more layers can determine the optical distance between the sample well and the sensor. In some embodiments, the thickness of one or more layers can determine the optical distance between the sample well and the sensor in the pixel. Additionally or alternatively, the refractive index of the materials forming one or more layers of integrated device 25-200 can determine the optical distance between sample well 25-203 and sensor 25-275 in a pixel. can. Such optical distance between sample well and sensor in a pixel may be less than 1 mm, less than 100 μm (microns), less than 25 microns, and/or less than 10 microns. One or more layers are present between the sample well layer 25-201 and the waveguide layer 25-220 to improve the coupling of excitation energy from the waveguide 25-220 to the sample well 25-203. may Although the integrated device 25-200 shown in FIG. 25 illustrates only a single layer 25-210, multiple layers may be formed between the sample well 25-203 and the waveguide 25-220. good. Layers 25-210 may be formed with optical properties that improve the coupling of excitation energy from waveguides 25-220 to sample wells 25-203. Layers 25-210 may be configured to reduce scattering and/or absorption of excitation energy and/or increase emission from samples in sample wells 25-203. Layers 25-210 may, according to some embodiments, be optically transparent so that light may travel to and from sample wells 25-203 with little attenuation. In some embodiments, dielectric materials may be used to form layers 25-210. In some embodiments, the excitation energy coupling component in layer 25-210 and/or at the interface between layer 25-210 and sample well layer 25-201 is coupled from waveguide 25-220 to sample well 25-203. may be provided to improve the coupling of the excitation energy to the As an example, the energy collection component 25-215 formed at the interface between the sample well layer 25-201 and the layer 25-210 directs the coupling of excitation energy from the waveguide 25-220 to the sample well 25-203. may be configured to improve The energy collection components 25-215 are optional, and in some embodiments, the configuration of the waveguides 25-220 and sample wells 25-203 can provide adequate excitation energy without the presence of the excitation energy collection components 25-215. coupling can be made possible.

Luminescent light or energy emitted from samples in sample wells 25-203 can be transmitted to sensors 25-275 in a variety of ways, some examples of which are described in detail below. Some embodiments include optical components to increase the likelihood that light of a particular wavelength will be directed to an area or portion of sensor 25-275 dedicated to detecting light of this particular wavelength. may be used. Sensors 25-275 may include multiple portions for simultaneously detecting different wavelengths of light that may correspond to emissions from different luminescent markers.

There may be one or more layers between sample well 25-203 and sensor 25-275 that may be configured to improve the collection of emissions from sample well 25-203 to sensor 25-275. . A luminescence directing component may be at the interface between sample well layer 25-201 and layer 25-210. The energy collection components 25-215 may focus the radiant energy onto the sensors 25-275 and may also or alternatively spatially separate radiant energies having different characteristic energies or wavelengths. Such energy collection components 25-215 may include grating structures for directing emitted light to sensors 25-275. In some embodiments, the lattice structure may be a series of concentric rings or "bull's eye" lattice structure configurations. A concentric grid may protrude from the bottom surface of the sample well layer 25-201. A circular grating can act as a plasmonic element that can be used to reduce the spread of signal light and direct the signal light to the associated sensor 25-275. Such a bull's eye grating can direct emitted light to the sensors 25-275 more efficiently.

Layers 25-225 may be formed adjacent to the waveguide. The optical properties of layers 25-225 may be selected to improve collection of emitted light from the sample wells to sensors 25-275. In some embodiments, layers 25-225 may be dielectric materials. A baffle may be formed between the sample well layer 25-201 and the sensor 25-275. Baffles 25-240 may be configured so that sensors 25-275 receive emission corresponding to sample wells 25-203 and reduce emission and reflected/scattered excitation from other sample wells. Filtering elements 25-260 may be arranged and configured to reduce the excitation energy reaching sensors 25-275. In some embodiments, filtering elements 25-260 may include filters that selectively transmit radiant energy of one or more markers used to label the sample. In embodiments with arrays of sample wells and arrays of sensors, where each sample well has a corresponding sensor, the baffle corresponding to each sample well receives from other sample wells collected by the sensor corresponding to the sample well. It can be configured to reduce luminescence and reflected and/or scattered excitation light.

One or more layers may be formed between the waveguides 25-220 and the sensors 25-275 to reduce the transmission of excitation energy to the sensors. In some embodiments, filtering elements may be formed between waveguides 25-220 and sensors 25-275. Such filtering elements may be configured to reduce the transmission of excitation energy to sensors 25-275 while allowing emissions from sample wells to be collected by sensors 25-275.

The radiant energy may be detected by sensors 25-275 and converted into at least one electrical signal. The electrical signals may be transmitted along one or more column or row conductive lines (not shown) to integrated electronics on substrate 25-200 for subsequent signal processing.

The above description of FIG. 25 is an overview of some components of instruments according to some embodiments. In some embodiments, one or more elements of FIG. 25 may be absent or in different locations. The components of integrated device 25-200 and excitation sources 25-230 are described in more detail below.

A. Excitation Source Coupling Region The integrated device has an excitation source coupling region configured to couple with an external excitation energy source and direct excitation to at least one pixel in the pixel area of the integrated device. can be done. The excitation source coupling region can include one or more structures configured to couple light into at least one waveguide. Any suitable mechanism for coupling excitation energy into the waveguide may be used.

An excitation source coupling region of an integrated device may include structural components configured to couple with an external excitation source. The integrated device may include a grating coupler configured to couple with an external excitation source located proximate to a surface of the integrated device and direct light into at least one waveguide of the integrated device. good. Grating coupler features such as size, shape, and/or grating configuration can be configured to improve coupling of excitation energy from the excitation source into the waveguide. A grating coupler can include one or more structural components in which the spacing between the structural components can serve to propagate light. One or more dimensions of the grating coupler can provide the desired coupling of light having certain characteristic wavelengths.

An integrated device can also include a waveguide having a tapered region at one end of the waveguide. A dimension or dimensions of the waveguide perpendicular to the direction of light propagation in the waveguide may be greater at one end of the waveguide, forming a tapered region of the waveguide. In some embodiments, the tapered region of the waveguide is perpendicular to the propagation of light and on the surface of the integrated device, larger at one end of the waveguide and smaller along the length of the waveguide. It may have a dimension parallel to it. In embodiments that include a grating coupler, the tapered region may be positioned proximate the grating coupler such that the larger end of the tapered region is located closest to the grating coupler. The tapered region enhances the coupling of light between the grating coupler and the waveguide by enlarging one or more dimensions of the waveguide to allow for improved mode overlap between the waveguide and the grating coupler. It may be of size and shape for improvement. In this way, an excitation source located close to the surface of the integrated device can couple light into the waveguide through the grating coupler. Such a combination of grating coupler and waveguide taper may allow more tolerance in the alignment and positioning of the excitation source with respect to the integrated device.

The grating coupler may be placed in a region of the integrated device outside the pixels of the integrated device. On the surface of the integrated device, the pixel sample well may occupy an area of the surface remote from the excitation source coupling region. An excitation source located proximate to the surface of the excitation source coupling region can be coupled with the grating coupler. The sample well may be located away from the excitation source coupling region to reduce interference of light from the excitation source to pixel performance. The grating coupler of the integrated device may be formed in one or more layers of the integrated device that contain the waveguides. Thus, the excitation source coupling region of the integrated device can include the grating coupler in the same plane of the integrated device as the waveguide. Grating couplers may be configured for a particular set of beam parameters, including beam width, angle of incidence, and/or polarization of incident excitation energy.

A cross-sectional view of integrated device 26-200 is shown in FIG. Integrated device 26-200 includes at least one sample well 26-222 formed in layer 26-223 of integrated device 26-200. Integrated device 26-200 includes grating coupler 26-216 and waveguide 26-220 formed in substantially the same plane of integrated device 26-200. In some embodiments, grating couplers 26-216 and waveguides 26-220 may be formed from the same layer of integrated device 26-200 and include the same materials. The source coupling region 26-201 of the integrated device 26-200 includes a grating coupler 26-216. As shown in FIG. 26, the sample well 26-222 is located on the surface of the integrated device 26-200 outside the excitation source coupling region 26-201. An excitation source 26-214 positioned relative to the integrated device 26-200 can provide incident excitation energy to the surface 26-215 of the integrated device 26-200 within the excitation source coupling region 26-201. can. By placing the grating coupler 26-216 within the excitation source coupling region 26-201, the grating coupler 26-216 couples the excitation energy from the excitation source 26-214 and directs the excitation energy to the waveguide 26-220. can be coupled to Waveguides 26-220 are configured to propagate excitation energy proximate one or more sample wells 26-222.

A grating coupler may be formed from one or more materials. In some embodiments, a grating coupler may include alternating regions of different materials along a direction parallel to the propagation of light in the waveguide. As shown in FIG. 26, grating coupler 26-216 includes a structure surrounded by material 26-224. The material or materials forming the grating coupler may have a refractive index or indices suitable for coupling and propagating light. In some embodiments, a grating coupler may include a structure formed from one material surrounded by a material with a higher index of refraction. As an example, a grating coupler may include a structure formed from silicon nitride and surrounded by silicon dioxide.

Any suitable dimensions and/or intergrating spacings can be used to form the grating couplers. Grating couplers 26-216 have dimensions perpendicular to the propagation of light through the waveguide, such as about 50 nm, about 100 nm, about 150 nm, or about 200 nm along the y-direction as shown in FIG. can have The spacing between structures of the grating coupler along the direction parallel to the propagation of light in the waveguide, for example along the z-direction as shown in FIG. 26, can have any suitable distance. . The spacing may be about 300 nm, about 350 nm, about 400 nm, about 420 nm, about 450 nm, or about 500 nm. In some embodiments, the intergrating spacing may be variable within the grating coupler. Grating couplers 26-216 are one or more substantially parallel to surfaces 26-215 of integrated device 26-200 that provide adequate area for coupling with external excitation sources 26-214. can have dimensions. The area of grating coupler 26-216 may match one or more dimensions of the light beam from excitation source 26-214 such that the beam overlaps with grating coupler 26-215. A grating coupler may have an area configured for a beam diameter of about 10 microns, about 20 microns, about 30 microns, or about 40 microns.

The integrated device can include a layer formed on the side of the grating coupler opposite the excitation source that is configured to reflect light. The layer can reflect excitation energy passing through the grating coupler towards the grating coupler. Including layers in an integrated device can improve the efficiency of coupling excitation energy into the waveguide. An example of a reflective layer is layers 26-218 of integrated device 26-200 shown in FIG. Layer 26-218 is disposed within excitation source coupling region 26-201 of integrated device 26-200 and is configured to reflect light toward grating coupler 26-216. Layers 26-218 are formed proximate the side of grating coupler 26-216 opposite the incident excitation energy from excitation source 26-214. Placing the layers 26-218 outside the pixels of the integrated device 26-200 may reduce the interference of the layers 26-218 with the performance capabilities of the pixels. Layers 26-218 may comprise any suitable material. Layers 26-218 may be substantially reflective to one or more excitation energies. In some embodiments, this layer may include Al, AlCu, and/or TiN.

B. Waveguides The integrated device may include one or more waveguides arranged to apply a desired amount of excitation energy to one or more sample wells of the integrated device. A waveguide is positioned proximate the one or more sample wells such that a portion of the excitation energy is coupled into the one or more sample wells as the excitation energy propagates along the waveguide. The waveguide couples excitation energy to multiple pixels and can act as a bus waveguide. For example, a single waveguide can apply excitation energy to a column or row of pixels in an integrated device. In some embodiments, a waveguide may be configured to propagate excitation energy having multiple characteristic wavelengths. A pixel of the integrated device can include additional structures (eg, microcavities) configured to direct excitation energy from the waveguide to the vicinity of the sample well. In some embodiments, the waveguide may have an optical mode with an evanescent tail configured to extend into the sample well and/or regions near the sample well. Additional energy coupling structures located near the sample well can couple energy from the evanescent tail to the sample well.

One or more dimensions of the waveguide of the integrated device may provide desired propagation of excitation energy along the waveguide and/or to one or more sample wells. A waveguide can have a dimension perpendicular to the propagation of light and parallel to the plane of the waveguide, which can be considered a cross-sectional width. The waveguide has a cross-sectional width of about 0.4 microns, about 0.5 microns, about 0.6 microns, about 0.65 microns, about 0.8 microns, about 1 micron, or about 1.2 microns. may A waveguide can have a dimension perpendicular to the propagation of light and perpendicular to the plane of the waveguide, which can be considered a cross-sectional height. The waveguide has a cross-sectional height of about 0.05 microns, about 0.1 microns, about 0.15 microns, about 0.16 microns, about 0.17 microns, about 0.2 microns, or about 0.3 microns may have In some embodiments, the waveguide has a cross-sectional width that is greater than its cross-sectional height. The waveguides have a distance D between the sample wells 26-222 and the waveguides 26-220, such as the distance D shown in FIG. 26, which is about 0.3 microns, 0.5 microns, or about 0.7 microns. It may be located at a particular distance from one or more sample wells in the integrated device.

In an exemplary embodiment, the waveguide may have a cross-sectional width of about 0.5 μm and a cross-sectional height of about 0.1 μm, and may be positioned about 0.5 μm below the sample well layer. . In another exemplary embodiment, the waveguide may have a cross-sectional width of approximately 1 μm and a cross-sectional height of 0.18 μm, and may be positioned 0.3 μm below the sample well layer.

The waveguide may be dimensioned to support a single transverse radiation mode, or it may be dimensioned to support multiple transverse radiation modes. In some embodiments, one or more dimensions of the waveguide may act so that the waveguide maintains only a single transverse mode and may selectively propagate TE or TM polarized modes. . In some implementations, the waveguide may have a highly reflective section formed at its end such that it supports longitudinal vertical modes within the waveguide. By supporting one mode, the waveguide may have reduced modal interference effects from cross-coupling of modes with different propagation constants. In some embodiments, the highly reflective section comprises a single highly reflective surface. In other embodiments, the highly reflective section comprises a plurality of reflective structures that collectively provide high reflectivity. A waveguide may be configured to split excitation energy from a single excitation source with higher output intensity using a waveguide beam splitter that produces multiple excitation energy beams from a single excitation source. Such beamsplitters may include evanescent coupling mechanisms. Additionally or alternatively, photonic crystals may be used in waveguide structures to improve excitation energy propagation and/or in materials surrounding the waveguides to reduce excitation energy scattering.

With respect to other components in the pixel of the integrated device, the position and arrangement of the waveguides improve the coupling of excitation energy towards the sample well, improve the collection of radiant energy by the sensor, and/or introduce by the excitation energy. can be configured to reduce the signal noise received. The waveguide may be sized and/or positioned with respect to the sample well to reduce interference of excitation energy propagating in the waveguide with radiant energy emanating from the sample well. The positioning and alignment of the waveguides within the integrated device can depend on the refractive indices of the waveguides and the materials surrounding the waveguides. For example, the waveguide perpendicular to the direction of light propagation along the waveguide, and the dimensions in the plane of the waveguide, allow a substantial amount of radiant energy from the sample well to propagate to the pixel's sensor. may be increased to allow passage of In some implementations, the distance between the sample well and the waveguide and/or the waveguide thickness are selected to reduce reflections from one or more interfaces between the waveguide and surrounding materials. may According to some embodiments, the reflection of radiant energy by the waveguide is less than about 5% in some embodiments, less than about 2% in some embodiments, and even less than about 1% in some embodiments. % can be reduced.

The ability of a waveguide to propagate excitation energy can depend on both the material of the waveguide and the material surrounding the waveguide. Thus, a waveguide structure may include core material, such as waveguides 26-220, and cladding material, such as regions 26-224, as illustrated in FIG. Both the waveguide and the surrounding material can allow excitation energy having a characteristic wavelength to propagate through the waveguide. The material, either the waveguide or the surrounding material, may be selected for a particular refractive index or combination of refractive indices. The waveguide material may have a lower refractive index than the waveguide surrounding material. Exemplary waveguide materials include silicon nitride (Si _x N _y ), silicon oxynitride, silicon carbide, tantalum oxide (TaO ₂ ), aluminum dioxide. Exemplary waveguide surrounding materials include silicon dioxide (SiO ₂ ) and silicon oxide. The waveguide and/or surrounding material can include one or more materials. In some examples, a desired index of refraction for the waveguide and/or surrounding material may be obtained by forming the waveguide and/or surrounding material to include more than one material. In some embodiments, the waveguide comprises silicon nitride and the surrounding material comprises silicon dioxide.

Splitting the waveguide from a single waveguide into multiple waveguides can allow excitation energy to reach multiple columns or rows of sample wells in an integrated device. An excitation source may be coupled to an input waveguide, which may be split into multiple output waveguides, each output waveguide applying excitation energy to a column or row of sample wells. Any suitable technique for splitting and/or combining waveguides may be used. Such waveguide splitting and/or combining methods may include star splitters or couplers, Y splitters, and/or evanescent couplers. Additionally or alternatively, a multi-mode interference splitter (MMI) may be used to split and/or combine the waveguides. One or more of these waveguide splitting and/or combining methods can be used to direct the excitation energy to the sample well.

C. Sample Wells According to some embodiments, sample wells 27-210 may be formed in one or more pixels of an integrated device. A sample well was formed in the surface of substrate 27-105, and sample 27-101 was formed on the surface of the substrate, as depicted in FIGS. 27A and 27B illustrating a single pixel 27-100 of the integrated device It may comprise small volumes or regions arranged to allow diffusion into and out of the sample well from the deposited specimen. In various embodiments, sample wells 27-210 may be arranged to receive excitation energy from waveguides 27-240. A sample 27-101 that diffuses into the sample well can be temporarily or permanently held within the excitation region 27-215 of the sample well by an adhesive 27-211. In the excitation region, the sample can be excited by excitation energy (eg, excitation radiation 27-247) and then emit radiation that can be observed and evaluated to characterize the sample.

In further details of operation, at least one sample 27-101 to be analyzed may be introduced into the sample wells 27-210 from, for example, a specimen (not shown) containing a fluid suspension of the sample. The excitation energy from the waveguides 27-240 is at least contained in the sample or at least one marker or tag associated with the sample while the sample is within the excitation region 27-215 in the sample well. One marker can be excited. According to some embodiments, markers may be luminescent molecules (eg, fluorophores) or quantum dots. In some implementations, there may be more than one marker used to analyze the sample (e.g., J. Eid, et al., incorporated by reference in its entirety). in "Real-Time DNA Sequencing from Single Polymerase Molecules", Science 323, p.133 (2009). different markers and tags). During and/or after excitation, the sample or marker may emit radiant energy. When multiple markers are used, they may emit with different energy profiles and/or with different temporal profiles, including different lifetimes. Radiant energy from the sample well can be radiated or otherwise transferred to sensors 27-260 where the radiant energy is detected and converted into electrical signals that can be used to characterize the sample.

According to some embodiments, sample wells 27-210 may be partially enclosed structures as depicted in FIG. 27B. In some implementations, sample wells 27-210 comprise submicron-sized holes or gaps (characterized by at least one transverse dimension D _sw ) formed in at least one layer of material 27-230. In some cases, holes may be referred to as "nano-apertures." The transverse dimension of the sample well may be from about 20 nanometers to about 1 micron according to some embodiments, although larger and smaller sizes may be used in some implementations. The volume of sample wells 27-210 may be from about 10 ⁻²¹ liters to about 10 ⁻¹⁵ liters in some implementations. A sample well may be formed as a waveguide that may or may not support a propagating mode. A sample well may be formed as a waveguide that may or may not support a propagating mode. In some embodiments, the sample well may be formed as a zero-mode waveguide (ZMW) having a cylindrical shape (or similar shape) with a diameter (or maximum transverse dimension) D _sw . ZMWs may be formed in a single metal layer as nanoscale holes that do not support propagating optical modes through the holes.

Due to the small volume of sample wells 27-210, a single sample event (e.g., single single-molecule events) can be enabled. For example, micromolar concentrations of sample may be present in a specimen placed in contact with an integrated device, but at the pixel level only about one sample (or single molecule event) is in the sample well at any given time. can be Statistically, some sample wells may contain samples and some may not contain more than one sample. However, a significant number of sample wells may contain a single sample (eg, at least 30% in some embodiments) so that single molecule analysis can be performed on multiple pixels simultaneously. Because single-molecule or single-sample events can be analyzed at each pixel, the integrated device allows detection of rare events that would otherwise not be noticed with ensemble averaging.

A sample well has a transverse dimension D _sw in some embodiments from about 500 nanometers (nm) to about 1 micron, in some embodiments from about 250 nm to about 500 nm, in some embodiments from about 100 nm to about 250 nm. , and even from about 20 nm to about 100 nm in some embodiments. According to some implementations, the sample well has a transverse dimension of about 80 nm to about 180 nm, or about 1/4 to 1/8 of the excitation or emission wavelength. According to other implementations, the sample well has a transverse dimension of about 120 nm to about 170 nm. In some embodiments, the depth or height of sample wells 27-210 may be from about 50 nm to about 500 nm. In some implementations, the depth or height of sample wells 27-210 may be from about 80 nm to about 250 nm.

Sample wells 27-210 with sub-wavelength transverse dimensions can improve the operation of pixels 27-100 of integrated devices in at least two ways. For example, incident excitation energy into the sample well from the opposite side of the specimen may couple into the excitation regions 27-215 with exponentially decreasing forces and not propagate through the sample well into the specimen. As a result, the excitation energy is increased in the excitation region where it excites the sample of interest and is reduced in the specimen where it excites other samples contributing to background noise. Also, because radiation propagating upward through the sample well is highly suppressed, radiation from the sample held at the bottom of the well (eg, closer to the sensors 27-260) is preferably directed toward the sensor. be done. Both of these effects can improve the signal-to-noise ratio in a pixel. The inventors recognize several aspects of sample wells that can be improved to further increase the signal-to-noise level at the pixel. These aspects relate to sample well geometry and structure, and also to adjacent optical and plasmonic structures (described below) that assist in coupling excitation energy into and radiation emitted from the sample well.

According to some embodiments, sample wells 27-210 may be formed as nano-apertures that are configured not to support propagation modes for particular wavelengths of interest. In some examples, a nanoaperture is constructed in which all modes are below the threshold wavelength, and the aperture may be a sub-cutoff nanoaperture (SCN). For example, sample wells 27-210 may comprise cylindrical holes or perforations in the conductive layer. The cross-section of the sample well need not be round, and may be oval, square, rectangular, or polygonal in some embodiments. Excitation energy 27-247 (eg, visible or near-infrared radiation) may be defined by walls 27-214 of the sample well at the first end of the well, as depicted in FIG. 27B. The sample well can be entered through 212 . When configured as an SCN, the excitation energy can decay exponentially along the length of the nanoaperture (eg, towards the specimen). In some implementations, the waveguide may have an SCN for radiation emitted from the sample, but not an SCN for excitation energy. Since the excitation energy can have a shorter wavelength than the emitted radiation, the apertures and waveguides formed by, for example, the sample well can be large enough to support the propagating modes for the excitation energy. Longer wavelength radiation can exceed the cutoff wavelength of the propagating mode in the waveguide. According to some embodiments, the sample wells 27-210 have the maximum intensity of the excitation energy localized in the sample well excitation region 27-215 at the entrance of the sample well 27-210 (eg, An SCN for the excitation energy may be provided (localized near the interface between layers 27-235 and 27-230 as depicted). Such localization of excitation energy improves the localization of radiant energy from the sample and can limit the observed radiation emitted from a single sample (eg, single molecule).

Pixels 27-100 may include additional structures, according to some embodiments. For example, pixel 27-100 can include one or more excitation coupling structures 27-220 that affect the coupling of excitation energy to the sample within the sample well. The pixels can also include radiation directing structures 27-250 that affect directing radiant energy from the sample in the sample well to the sensor 27-260.

Other sample well structures have been developed and tested by the inventors to improve the intensity of the excitation energy localized to the sample well. FIG. 27C depicts an embodiment of a sample well that includes cavities or divots 27-216 at the excitation end of the sample well. Adding divots 27-216 to the sample well allows the sample to move to regions of higher excitation intensity, according to some embodiments. In some implementations, the shape and structure of the divot changes the local excitation field (e.g., due to the difference in refractive index between layers 27-235 and the fluid in the sample well), causing the excitation energy in the divot to change. Strength can be further increased. Divots 27-216 may be formed in layers 27-235 such that a portion of the sample volume occupying sample wells 27-214 and divots 27-216 are surrounded by the material forming layers 27-216. .

The divot can have any suitable shape. The divot can have a transverse shape substantially equal to the transverse shape of the sample well, eg, circular, oval, square, rectangular, polygonal, and the like. In some embodiments, the sidewalls of the divot may be substantially straight and vertical like the walls of the sample well. In some implementations, the sidewalls of the divot may be slanted and/or curved as depicted in the figures. The transverse dimension of the divot may be about the same size as the transverse dimension of the sample well in some embodiments, smaller than the transverse dimension of the sample well in some embodiments, or may be greater than the transverse dimension of the sample well. The divots 27-216 may extend from about 10 nm to about 200 nm beyond the sample well layer 27-230. In some implementations, the divots may extend about 50 nm to about 150 nm beyond the sample well layers 27-230. In some embodiments, the divots may extend about 150 nm to about 250 nm beyond the sample well layers 27-230. By forming divots, excitation regions 27-215 can extend outside the sample well as depicted in FIG. 27C.

Some embodiments relate to an integrated device having a sample well with a divot positioned proximate to a waveguide. FIG. 28 shows an integrated device having sample wells 28-632 formed in layers 28-630 and 28-636. Layers 28-630 may be metal layers and may include one or more metals (eg, Al). Layers 28-636 may function as dielectric layers and may include one or more dielectric materials (eg, silicon dioxide). Sample wells 28-632 may have variable dimensions in directions parallel to layers 28-630 and/or layers 28-636. The sample well 28-632 may have a dimension D2 along the z-direction within at least the layer 28-630 of the integrated device, and in some embodiments the dimension D2 is the diameter of the sample well 28-632. may be considered. Dimension D2 of sample well 28-632 may be about 700 nm, about 800 nm, about 900 nm, about 1 micron, or about 1.1 microns. The sample well 28-632 may have a dimension D1 along the z-direction within the layer 28-636 of the integrated device, which in some embodiments is considered the diameter at the surface of the sample well 28-632. may Dimension D1 may be about 100 nm, about 150 nm, about 200 nm, or about 250 nm. A surface of sample well 28-632 having dimension D1 is positioned dimension d1 along the x-direction from waveguide 28-634. Placing sample well 28-632 closer to waveguide 28-634 by distance d1 may allow for improved coupling of excitation energy from waveguide 28-634 to sample well 28-632. Dimension d1 may be about 50 nm, about 100 nm, about 150 nm, about 200 nm, or about 250 nm.

D. Coupling Excitation Energy to Sample Wells Coupling excitation energy to one or more sample wells of an integrated device can occur by one or more techniques. As discussed above, in some embodiments the waveguide is arranged to couple with an excitation source for one or more sample wells. As the excitation energy propagates along the waveguide, a portion of the excitation energy can be coupled to one or more sample wells by various optical coupling methods. For example, a waveguide can direct excitation energy in substantially one direction, forming an evanescent wave or tail perpendicular to this one direction, and in some examples can be located outside the waveguide structure. . Such evanescent tails can direct some of the excitation energy into one or more sample wells. In some embodiments, the sample well layer may be designed and configured to direct excitation energy to localized regions within the sample well. The sample well may be configured to retain the sample within a localized region of the sample well such that excitation energy is directed to the sample.

Additionally, one or more components may be formed into an integrated device to improve or enhance the coupling of excitation energy to the sample well. These additional components may be formed within the pixel and may provide coupling of excitation energy from the waveguide to the pixel and sample well. One or more components located within the pixel can serve to channel some of the excitation energy from the waveguide into the pixel. Such components can include optical structures such as grating structures, scattering structures, microcavities and/or nanoantennas. The characteristics or configuration of one or more of these components can be selected to couple a particular amount of excitation energy to each sample well within a column or row of sample wells. A waveguide configured to provide excitation energy to a column of pixels is coupled to a component in each pixel region to provide a portion of the excitation energy to each pixel in the column of pixels. can be done. When a waveguide is configured to direct excitation energy from an excitation source to one or more pixels, the waveguide may be referred to as a bus waveguide.

A component placed adjacent to the sample well can improve the coupling of excitation energy from the waveguide to the sample well. Such components may be referred to as taps and/or microcavities. The microcavity can divert some of the excitation energy from the waveguide so that the excitation energy reaches the sample well. One or more microcavities can be used to couple excitation energy to the sample well. One or more microcavities can reduce the loss of excitation energy from the waveguide, including metal losses. One or more microcavities can act as lenses that focus the excitation energy into the sample well. In some embodiments, one or more microcavities can improve the guidance of luminescence from the markers in the sample wells to the sensor. A microcavity can have a cylindrical, convex, concave, rectangular, spherical, or elliptical configuration, or any other suitable shape. Microcavities can be formed from any suitable material. In some embodiments, the microcavities may comprise silicon nitride.

The one or more microcavities may overlap at least part of the waveguide that directs the excitation energy to the sample well when viewed from the top of the integrated device in which the sample well resides. The waveguide thickness may be configured to reduce excitation energy loss and improve excitation energy coupling to the one or more microcavities. In some embodiments, the microcavities along the row of sample wells may have different coupling strengths between the waveguides for each sample well. The microcavity can increase the coupling along the propagation direction of the excitation energy to accommodate the power drop in the waveguide as the excitation energy is directed from the waveguide to each sample well. In some embodiments, one or more microcavities are adjacent to the sample well. There may be an offset distance between the location of the center of the sample well and the center of the microcavity. In other embodiments, one microcavity is below the sample well such that at least a portion of the sample well and a portion of the microcavity overlap when viewed from the top of the integrated device in which the sample well resides. located in

One or more dimensions of the waveguide of the integrated device may vary along the length of the waveguide in the direction of light propagation through the waveguide. By varying one or more dimensions along the waveguide, the coupling efficiency and substantial uniformity of the amount of excitation energy provided by the waveguide to multiple sample wells can be improved. In some embodiments, the cross-sectional width of the waveguides may vary along the columns or rows of pixels. The waveguide may include a taper such that the cross-sectional width of the waveguide decreases along the direction of propagation of excitation energy through the waveguide. In some embodiments, tapered waveguides may be configured for similar coupling efficiencies for a row of pixels where the pixels in the row include microcavities and sample wells. The combination of varying one or more dimensions of the tapered waveguide and/or microcavity reduces the power of the excitation energy along the length of the waveguide as it is coupled into each sample well in the array. can be adjusted.

In some embodiments, the waveguide can be coupled to the sample well by an evanescent field. In some embodiments, a bull's eye grating structure with concentric grating rings located close to the sample well can improve the coupling of excitation energy from the waveguide to the sample well. In some embodiments, the waveguide may include a region of reduced cross-sectional width in the vicinity of the sample well such that evanescent fields from excitation energy propagating in the waveguide are coupled to the sample well. . For a single column of pixels, the waveguide may include multiple regions of reduced cross-sectional width along the length of the waveguide to improve coupling uniformity and efficiency between sample wells in the column. good. As such, the waveguide can be viewed as having a tapered section at a particular location along the waveguide.

Layers of an integrated device with one or more sample wells may interfere with the propagation of light through the waveguide. In some embodiments, the sample well layer is formed of metal (eg, aluminum). It may be desirable to place the sample well layer at a particular distance from the waveguide so as to reduce and improve device performance losses. These approaches may allow the desired performance to be achieved by placing the sample well layer at a particular distance from the waveguide, while allowing the sample well within the layer to receive a sufficient amount of excitation energy. do.

E. Directing Radiant Energy to the Sensor The integrated device includes one or more components positioned between the pixel sample well and the sensor to improve the collection of emitted light by the sensor from the sample in the sample well. It's okay. Such components can improve the signal-to-noise ratio of luminescent signal to background signal to improve detection of luminescent markers. Some components can direct emission from a sample well to sensors in corresponding pixels. In some embodiments, the component may provide both suitable coupling of excitation energy to the sample well and coupling of emission from the sample well. Other components (eg, filters) can reduce excitation energy and other light not associated with the sample and/or markers that contribute to the signal acquired by the sensor.

A bull's eye grid can be formed from concentric grid rings around the sample well. A bull's eye grating can be coupled with the sample well to improve the propagation of luminescence from the sample well. A bull's eye grating structure can direct light emission to the sensor in the pixel with the sample well. In some embodiments, the effective diameter of emitted light directed by the bull's eye grating is about 5 microns. In some embodiments, a waveguide for propagating excitation energy to the sample well and one or more microcavities provided for coupling the sample well also direct emission from the sample well to the sensor. can be done.

A baffle with a gap centered on the sensor may be formed between the sample well and the sensor. As shown in FIG. 26, baffle layer 26-226 is positioned between sample well 26-22 and sensor layer 26-230. Baffles in pixels can be designed to suppress the collection of non-luminous energy for the pixel. A baffle associated with a sample well and sensor reduces emission from the sample well while reducing emission, excitation energy, and other energy from neighboring pixels not associated with emission from the sample well associated with the sensor. can be allowed to reach the sensor. The dimensions of the baffle gap may be configured such that emission is directed by the bull's eye on the same pixel. As shown in FIG. 26, baffles 26-226 have gaps along the z-direction to pass emitted light to sensors located in layers 26-230. Baffle materials may be selected for specific optical properties, such as reducing transmission of specific light wavelengths or energies at specific angles of incidence. In some embodiments, the baffle may be formed from multiple layers of materials having different refractive indices. The baffle may include one or more layers of silicon, silicon nitride (Si3N4), silicon, titanium nitride (TiN), and aluminum (Al). Layers configured to form baffles may have a cross-sectional height of about 20 nm, about 20 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, or about 90 nm.

A filtering component may be formed between the waveguide and the sensor to reduce collection of excitation energy by the sensor. Any suitable method for filtering the excitation energy may be provided. Techniques for filtering excitation energy may include filtering based on one or more properties of light. Such properties may include wavelength and/or polarization. A filtering component can selectively suppress scattering of excitation energy while passing emission to the sensor. Layers 26-228 shown in FIG. 26 may include one or more filtering components described herein.

The integrated device may include wavelength filters configured to reflect light of one or more characteristic wavelengths and to allow transmission of light having different characteristic wavelengths. In some embodiments, the light reflected by the wavelength filter may have a shorter characteristic wavelength than the light transmitted by the wavelength filter. Such wavelength filters are because the excitation energy used to excite the marker typically has a characteristic wavelength shorter than the emission emitted by the marker in response to reaching an excited state with the excitation energy. It can reflect excitation energy and allow transmission of luminescence.

F. Sensors Any suitable sensor capable of obtaining time-bin information may be used for measurements that detect the lifetime of luminescent markers. For example, U.S. patent application Ser. No. 14/821,656, entitled "INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED PHOTONS," describes a photon arrival time determination. describes a sensor that can be used, which is incorporated by reference in its entirety. The sensors are aligned such that each sample well has at least one sensor area for detecting luminescence from the sample well. In some embodiments, the integrated device includes Geiger mode avalanche photodiode arrays and/or single photon avalanche diode arrays (SPAD).
photon avalanche diode array).

Described herein is the ability to precisely measure, or "time-bin", the timing of arrival of incident photons, e.g., in nucleic acid sequencing (e.g., DNA sequencing). is an integrated photodetector that can be used in a variety of applications such as. In some embodiments, the integrated photodetector can measure photon arrivals with nanosecond or picosecond resolution, facilitating time-domain analysis of incident photon arrivals.

Some embodiments are photodetectors that generate charge carriers in response to incident photons and can distinguish the timing at which charge carriers are generated by the arrival of incident photons with respect to a time reference (e.g., trigger event). It relates to an integrated circuit having In some embodiments, the charge carrier separation structure separates charge carriers generated at different times and includes one or more charge carrier storage areas (“bins”) that collect charge carriers generated within different time periods. directs the charge carriers into the Each bin stores charge carriers generated within a selected time interval. A readout of the charge stored in each bin can provide information on the number of photons that arrived within each time interval. Such integrated circuits may be used in any of a variety of applications such as those described herein.

Examples of integrated circuits with photodetection regions and charge carrier separation structures are described. In some embodiments, an integrated circuit may include an array of pixels, each pixel including one or more photodetector regions and one or more charge carrier separation structures, as discussed below. good. FIG. 29A shows a schematic representation of pixels 29-900 according to some embodiments. Pixels 29-900 are also referred to herein as photon absorption/carrier generation regions 29-902 (also called photodetection regions), carrier migration/trapping regions 29-906, "charge carrier storage bins" or simply "bins". It includes a carrier storage area 29-908 having one or more charge carrier storage areas and a readout circuit 29-910 for readout signals from the charge carrier storage bins.

The photon absorption/carrier generation region 29-902 may be a region of semiconductor material (eg, silicon) capable of converting incident photons into photogenerated charge carriers. Photon absorption/carrier generation regions 29-902 may be exposed to light and may receive incident photons. When photons are absorbed by the photon absorption/carrier generation regions 29-902, the regions can generate photogenerated charge carriers such as electron/hole pairs. Photogenerated charge carriers are also referred to herein simply as "charge carriers".

An electric field may be established within the photon absorption/carrier generation region 29-902. In some embodiments, the electric field may be "static," as distinguished from the changing electric field within the carrier migration/trapping region 29-906. The electric field within the photon absorption/carrier generation region 29-902 can include a lateral component, a longitudinal component, or both lateral and longitudinal components. A transverse component of the electric field may be in the downward direction in FIG. An electric field can be created in a variety of ways.

In some embodiments, one or more electrodes may be formed over the photon absorption/carrier generation regions 29-902. The electrodes can be energized to establish an electric field within the photon absorption/carrier generation region 29-902. Such electrodes are sometimes called "photogates". In some embodiments, the photon absorption/carrier generation regions 29-902 may be silicon regions that are completely depleted of charge carriers.

In some embodiments, the electric field within photon absorption/carrier generation region 29-902 may be established by a junction, such as a PN junction. The semiconductor material of the photon absorption/carrier generation region 29-902 forms a PN junction in an orientation and/or shape that produces an electric field that induces a force that drives the photogenerated charge carriers into the carrier migration/trapping region 29-906. may be doped as In some embodiments, the P monad of the PN junction diode may be connected to the monad that sets this voltage. Such diodes may be referred to as "pinned" photodiodes. A pinned photodiode can encourage carrier recombination at the surface by monadic attracting carriers that can set its voltage and reduce dark current. Photogenerated charge carriers that are desired to be trapped can pass under the recombination area of the surface. In some embodiments, the lateral electric field may be established using graded doping concentrations in the semiconductor material.

As illustrated in FIG. 29A, photons may be captured and charge carriers 29-901A (eg, electrons) may be generated at time t1. In some embodiments, the potential gradient causes photon absorption/carrier generation regions 29-902 and charge carriers 29-901A to move downward in FIG. 29A (as illustrated by the arrows shown in FIG. 29A). 2) may be established along carrier migration/trapping regions 29-906. In response to the potential gradient, charge carriers 29-901A move from a position at time t1 to a second position at time t2, a third position at time t3, a fourth position at time t4, and a fifth position at time t5. Can move to position. Charge carriers 29-901A thus migrate to carrier migration/trapping regions 29-906 in response to the potential gradient.

Carrier migration/trapping regions 29-906 may be semiconductor regions. In some embodiments, the carrier migration/trapping regions 29-906 may be protected from incident light (eg, by an overlying opaque material such as a metal layer). Except, it may be a semiconductor region of the same material as the photon absorption/carrier generation regions 29-902 (eg, silicon).

In some embodiments, and as discussed further below, a potential gradient is placed within and over the photon absorption/carrier generation region 29-902 and the carrier migration/trapping region 29-906. It may be established by electrodes. However, the techniques described herein are not limited to specific locations of the electrodes used to generate the potential gradient. Also, the techniques described herein are not limited to establishing a potential gradient using electrodes. In some embodiments, a potential gradient may be established using a spatially graded doping profile. Any suitable technique may be used to establish potential gradients that cause charge carriers to migrate along photon absorption/carrier generation regions 29-902 and carrier migration/trapping regions 29-906.

A charge carrier separation structure may be formed in the pixel to separate charge carriers generated at different times. In some embodiments, at least a portion of the charge carrier separation structure may be formed over the carrier migration/trapping regions 29-906. As described below, the charge carrier separation structure can include one or more electrodes formed over the carrier migration/trapping regions 29-906, the voltage of which is applied to the carrier migration/trapping regions 29-906. It can be controlled by a control circuit that changes the potential of -906.

The potential of carrier migration/trapping regions 29-906 can be varied to trap charge carriers. The potential gradient can be altered by varying the voltage of one or more electrodes over the carrier migration/trapping region 29-906 to create potential barriers that can confine carriers to predetermined spatial regions. For example, the voltage on the electrode overlying the dashed line of carrier migration/trapping region 29-906 in FIG. 29A creates a potential barrier along the dashed line of carrier migration/trapping region 29-906 in FIG. may be changed at time t5 to capture As shown in FIG. 29A, carriers captured at time t5 may be transferred to bin "bin 0" of carrier storage area 29-908. Transfer of carriers to the charge carrier storage bins is achieved by changing the potential within the carrier migration/trapping regions 29-906 and/or the carrier storage regions 29-908 (eg, by changing the voltage of electrodes covering these regions). to a charge carrier storage bin.

Varying the potential within a given spatial region of carrier migration/trapping region 29-906 at a particular time can allow localization of carriers generated by photon absorption that occurs within a particular time interval. By localizing the photogenerated charge carriers at different times and/or positions, the time at which the charge carriers were generated by photon absorption can be determined. In this sense, charge carriers can be "time-binned" by localizing the charge carriers at a particular point in time and/or space after the occurrence of a triggering event. The time binning of the charge carriers within a particular bin is the time at which the photogenerated charge carrier was generated by absorption of an incident photon, and thus also the "time bin" for the triggering event, the arrival of the incident photon that generated the photogenerated charge carrier. provide information about

FIG. 29B illustrates charge carrier trapping at different times and spaces. As shown in FIG. 29B, the voltage on the electrode overlying the dashed line of carrier migration/trapping region 29-906 creates a potential barrier along the dashed line of carrier migration/trapping region 106 in FIG. It may be changed at time t9 to acquire -901B. As shown in FIG. 29B, the captured carriers at time t9 can be transferred to bin "bin 1" of carrier storage area 29-908. Since charge carrier 29-901B is localized at time t9, it occurred at a different time (ie, time t6) than the photon absorption event (ie, t1) for carrier 29-901A captured at time t5. represents a photon absorption event.

Photons are captured in the photon absorption/carrier generation area 29-902 by taking multiple measurements and collecting charge carriers in charge carrier storage bins of the carrier storage area 29-908 based on the time the charge carriers are captured. can provide information about the time Such information can be useful in various applications as discussed above.

In some embodiments, the elapsed time may be different for each bin acquisition after the excitation pulse. For example, shorter time bins can be used to detect luminescence immediately after the excitation pulse, while longer time bins can be used at times away from the excitation pulse. By varying the time bin spacing, the signal-to-noise ratio for measuring the electrical signal associated with each time bin can be improved for a given sensor. Since the probability of a photon emission event is higher immediately after the excitation pulse, time bins within this time have shorter time intervals to account for the potential of more photons to detect. During longer times the probability of photon emission may be lower and the time bins to detect within this time may be longer to account for the lower potential number of photons. In some embodiments, time bins with significantly longer elapsed times may be used to distinguish multiple lifetimes. For example, a majority of the time bins may capture time intervals in the range of approximately 0.1-0.5 ns, while some time bins may capture time intervals of approximately 2-5 ns. The number of time bins and/or the time interval between each bin may depend on the sensor used to detect photons emitted from the sample object. Determining the time interval for each bin may include identifying the time interval required for the number of time bins provided by the sensor that distinguish the luminescent markers used to analyze the sample. be. The distribution of recorded histograms can be compared to known histograms of markers under similar conditions and time bins that identify the type of marker in sample wells. Different embodiments of the present application can measure the lifetime of markers, but differ in the excitation energy used to excite the markers, the number of sensor areas in each pixel, and/or the wavelengths detected by the sensors. there is a possibility.

III. Excitation Sources According to some embodiments, one or more excitation sources may be located external to the integrated device and arranged to deliver pulses of light to the integrated device with the sample well. good too. For example, U.S. Provisional Patent Application No. 62/164,485, entitled "PULSED LASER," filed May 20, 2015, and U.S. Provisional Patent Application No. 62/310,398 describes examples of pulsed laser light sources that can be used as excitation sources, each of which is incorporated by reference in its entirety. A pulse of light may be coupled to multiple sample wells and used, for example, to excite one or more markers within the wells. One or more excitation sources may deliver pulses of light of one or more characteristic wavelengths, according to some embodiments. In some cases, the excitation source may be packaged as an interchangeable module that is mounted or coupled into a base instrument into which the integrated device can be loaded. Energy from the excitation source may be delivered radiatively or non-radiatively to at least one sample well or to at least one sample of at least one sample well. In some embodiments, an excitation source with controllable intensity may be arranged to deliver excitation energy to multiple pixels of the integrated device. The pixels may be arranged in linear arrays (eg, rows or columns) or 2D arrays (eg, sub-areas of an array of pixels or a complete array of pixels).

Any suitable light source may be used as the excitation source. Some embodiments may use incoherent light sources and other embodiments may use coherent light sources. By way of non-limiting example, incoherent light sources according to some embodiments include different types of light emitting diodes (LED ). As a non-limiting example, coherent light sources according to some embodiments can be of different types of semiconductor lasers, such as vertical cavity surface emitting lasers (VCSELs), edge emitting lasers, and distributed feedback (DFB) laser diodes. May include a laser. Additionally or alternatively, a slab-coupled optical waveguide laser (SCOWL) or other asymmetric single-mode waveguide structure may be used. In some implementations, coherent light sources may comprise organic lasers, quantum dot lasers, and solid state lasers (eg, Nd:YAG or ND:glass lasers pumped by laser diodes or flash lamps). In some embodiments, a laser diode-pumped fiber laser may be used. A coherent light source may be passively mode-locked to generate ultrashort pulses. There may be multiple types of excitation sources for the array of pixels on the integrated device. In some embodiments, different types of excitation sources may be combined. The excitation source may be made according to conventional techniques used to make the selected type of excitation source.

By way of introduction and without limiting the invention, an example of a coherent light source arrangement is shown in FIG. 30A. This figure illustrates analytical instruments 30-100, which may include an ultrashort pulse laser excitation source 30-110 as an excitation source. The ultrashort pulse laser 30-110 includes a gain medium 30-105 (which can be a solid-state material in some embodiments), a pump source (not shown) that excites the gain medium, and at least two cavity mirrors 30-105. 102, 30-104 (defining the ends of the optical laser cavity). In some embodiments, there may be one or more additional optical elements within the laser cavity for beam shaping, wavelength selection, and/or pulse shaping purposes. In operation, the pulsed laser excitation source 30-110 emits ultrashort optical pulses 30 that circulate back and forth within the laser cavity between the cavity end mirrors 30-102, 30-104 and through the gain medium 30-105. -120 can be generated. One of the cavity mirrors 30-104 may partially transmit a portion of the circulating pulse, and the train of optical pulses 30-122 passes from the pulsed laser 30-110 to optical components and integrated devices, etc. It radiates to subsequent components 30-160. The emitted pulse can sweep the beam (indicated by the dashed line) characterized by the beam waist w. The measured temporal intensity profile of emitted pulse 30-122 may appear as shown in FIG. 30B.

In some embodiments, the peak intensity values of the emitted pulses may be approximately equal, and the profile may have a Gaussian temporal profile, although other profiles are possible, eg, a sech profile. In some cases the pulse may not have a symmetrical temporal profile and may have other temporal shapes. In some embodiments, gain and/or loss dynamics can result in pulses with asymmetric profiles. The duration of each pulse may be characterized by a full width at half maximum (FWHM) value, as shown in FIG. 30B. Ultrashort optical pulses may have FWHM values of less than 100 picoseconds.

Pulses emanating from a laser excitation source may be separated by regular intervals T. In some embodiments, T may be determined by the active gain and/or loss modulation rate in the laser. For mode-locked lasers, T may be determined by the round trip time between cavity end mirrors 30-102, 30-104. According to some embodiments, pulse separation time T may be between about 1 ns and about 100 ns. In some cases, pulse separation time T may be between about 0.1 ns and about 1 ns. In some implementations, the pulse separation time T may be between about 100 ns and about 2 μs.

In some embodiments, the optical system 30-140 may operate on a pulsed beam 30-122 from a laser excitation source 30-110. For example, the optics may comprise one or more lenses for reshaping the beam and/or changing the divergence of the beam. Reshaping the beam may comprise increasing or decreasing the value of the beam waist and/or changing the cross-sectional shape of the beam (eg, elliptical to circular, circular to elliptical, etc.). Altering the divergence of the beam may comprise converging or diverging the beam bundle. In some embodiments, optics 30-140 may include attenuators or amplifiers to change the amount of beam energy. In some cases, the optics may include wavelength filtering. In some implementations, the optical system may comprise pulse shaping elements, such as pulse stretchers and/or pulse compressors. In some embodiments, the optical system may comprise one or more nonlinear optical elements, such as saturable absorbers to reduce pulse length. According to some embodiments, optical system 30-140 may comprise one or more elements that alter the polarization of pulses from laser excitation source 30-110.

In some embodiments, optical system 30-140 is used to convert the output wavelength from pump source 30-110 to a shorter wavelength via frequency doubling or to a longer wavelength via parametric amplification. A nonlinear crystal may be provided. For example, the output of a laser may be frequency doubled in a nonlinear crystal (eg, periodically poled lithium niobate (PPLN)) or other non-poled nonlinear crystal. Such a frequency doubling process may allow more efficient lasers to produce wavelengths more suitable for excitation of the selected fluorophore.

The phrase "characteristic wavelength" or "wavelength" may refer to the central or dominant wavelength within the limited bandwidth of illumination produced by the excitation source. In some cases this may refer to the peak wavelength within the bandwidth of the illumination produced by the excitation source. A characteristic wavelength of the excitation source may be selected, for example, based on the selection of luminescent markers or probes used in the biological analysis device. In some embodiments, a characteristic wavelength of the excitation energy source is selected for direct excitation (eg, single-photon excitation) of the selected fluorophore. In some embodiments, a characteristic wavelength of the excitation source is selected for indirect excitation (eg, multiphoton excitation or harmonic conversion to a wavelength that provides direct excitation). In some embodiments, the excitation radiation may be produced by a light source configured to produce excitation energy at specific wavelengths for application to the sample well. In some embodiments, the characteristic wavelength of the excitation source may be smaller than the characteristic wavelength of the corresponding emission from the sample. For example, the excitation source may emit radiation having a characteristic wavelength between 500 nm and 700 nm (eg 515 nm, 532 nm, 563 nm, 594 nm, 612 nm, 632 nm, 647 nm). In some embodiments, the excitation source may provide excitation energy centered at two different wavelengths, such as 532 nm and 593 nm, for example.

In some embodiments, a pulsed excitation source may be used to excite the luminescent marker in order to measure the radiative lifetime of the luminescent marker. This can be useful for identifying luminescent markers based on their radiative lifetime rather than their emission color or wavelength. As an example, a pulsed excitation source can periodically excite a luminescent marker to generate and detect subsequent photon emission events used to determine the lifetime of the marker. A lifetime measurement of a luminescent marker is possible when the excitation pulse from the excitation source transitions from a peak pulse power or intensity to a lower (e.g., nearly extinguished) power or intensity for a time period shorter than the lifetime of the luminescent marker. can be This can be beneficial if the excitation pulse is terminated quickly to avoid re-excitation of the luminescent marker during the post-excitation phase when the lifetime of the luminescent marker is being evaluated. By way of example and not limitation, the pulse power may drop about 20 dB, about 40 dB, about 80 dB, or about 120 dB below the peak power after 250 picoseconds. In some implementations, the pulse power may drop about 20 dB, about 40 dB, about 80 dB, or about 120 dB below the peak power after 100 picoseconds.

A further advantage of using ultrashort excitation pulses to excite luminescent markers is to reduce photobleaching of the markers. Applying continuous excitation energy to the marker can fade and/or damage the luminescent marker over time. Even when the peak pulse power of the excitation source is well above the level at which continuous exposure would rapidly damage the marker, the use of ultrashort pulses reduces the time before the marker is damaged by the excitation energy and the useful measurement. number may increase.

When using a pulsed excitation source to discern the lifetime of a luminescent marker, the time between pulses of excitation energy should be as long as the longest lifetime of the marker in order to observe and evaluate the emission events after each excitation pulse. or longer. For example, the time interval T (see FIG. 30B) between excitation pulses can be longer than the radiative lifetime of any of the fluorophores examined. In this case, the subsequent pulse may not arrive before the excited fluorophore from the previous pulse has had a reasonable time to fluoresce. In some embodiments, the interval T determines the time between an excitation pulse that excites the fluorophore and the next photon emitted by the fluorophore after the end of the excitation pulse and before the next excitation pulse. must be long enough to

The interval T between excitation pulses should be long enough to observe the decay properties of the fluorophore, but it is also desirable that T be short enough to make many measurements in a short time. By way of example and not limitation, fluorophores used in some applications may have radiative lifetimes ranging from about 100 picoseconds to about 10 nanoseconds. Accordingly, excitation pulses used to detect and/or discern such lifetimes may have durations (FWHM) in the range of about 25 picoseconds to about 2 nanoseconds, and are about 20 MHz to about 1 GHz. may be provided with a pulse repetition rate in the range of .

More specifically, any suitable technique for modulating the excitation energy to generate a pulsed excitation source for lifetime measurements may be used. Direct modulation of an excitation source, such as a laser, can involve modulating the electrical drive signal of the excitation source so that the emitted power is in the form of pulses. The input power of the light source, including the optical pump power, and the excited state carrier injection and/or carrier removal from a portion of the gain region are modulated to affect the gain of the gain medium for pumping through dynamic gain shaping. It may allow the formation of pulses of energy. Additionally, the quality (Q) factor of the optical resonator may be modulated by various means for shaping pulses using Q-switching techniques. Such Q-switching techniques may be active and/or passive. Longitudinal modes of the laser's resonant cavity may be phase-locked to produce a train of pulses of emitted light through mode-locking. Such mode-locking techniques may be active and/or passive. The laser cavity may comprise a separate absorbing section that allows modulation of the carrier density and control of absorption losses in that section, providing an additional mechanism for shaping the excitation pulse. In some embodiments, an optical modulator may be used to modulate a beam of continuous wave (CW) light into pulses of excitation energy. In other embodiments, a signal sent to an acousto-optic modulator (AOM) coupled to the excitation source is used to change the polarization, intensity, frequency, phase and/or polarization of the output light to Pulsed excitation energy may be generated. AOMs may be used for continuous wave beam scanning, Q-switching, and/or mode-locking. Although the above techniques are described for generating a pulsed excitation source, any suitable method for generating a pulsed excitation source may be used to measure the lifetime of a luminescent marker.

In some embodiments, techniques for forming a pulsed excitation source suitable for lifetime measurements may involve modulation of an input electrical signal that drives photon emission. Some excitation sources (eg, diode lasers and LEDs) convert electrical signals, such as input current, into optical signals. Characteristics of the optical signal may depend on characteristics of the electrical signal. When generating a pulsed light signal, the electrical signal may change over time to generate a variable light signal. An optical signal having a particular waveform may be produced by modulating the electrical signal to have a particular waveform. The electrical signal may have a sinusoidal waveform with a certain frequency, and the resulting light pulses may occur within a time interval related to the frequency. For example, an electrical signal with a frequency of 500 MHz can produce an optical signal with a pulse every 2 nanoseconds. The combined beams produced by separate pulsed excitation sources may be similar or different from each other and may have relative optical path differences of less than 1 mm.

In some excitation sources, such as laser diodes, an electrical signal changes the carrier density, producing photons through recombination of electron-hole pairs. Carrier density is related to the optical signal, such that when the carrier density exceeds a threshold, a significant number of coherent photons are produced by stimulated emission. Current supplied to the laser diode can inject electrons or carriers into the device, thereby increasing the carrier density. If the carrier density is above the threshold, photons can be produced at a faster rate than the current supplied to the carriers, thus decreasing the carrier density below the threshold and reducing photon production. As photon production decreases, due to continued current injection and photon absorption, the carrier density starts to increase again and eventually increases above the threshold again. This cycle results in an oscillation of the carrier density near the threshold for photon generation, which gives rise to an oscillating optical signal. These dynamics, known as relaxation oscillations, can cause artifacts in the optical signal due to carrier density oscillations. When current is first supplied to the laser, there may be oscillations before the optical signal reaches a steady power due to oscillations in the carrier density. When forming a pulsed excitation source, carrier density oscillations can introduce artifacts in the pulsed light signal. Artifacts due to such relaxation oscillations broaden and/or create tails in the pulsed light signal, as the excitation signal can overlap with photons emitted by the luminescent marker, and are detected by such pulsed light sources. can limit the lifespan obtained.

In some embodiments, techniques of shortening the duration of the excitation pulse are used to reduce the excitation energy required to detect the luminescent marker, thereby reducing or delaying bleaching and other damage to the luminescent marker. can. Techniques that shorten the duration of the excitation pulse can also be used to reduce the power and/or intensity of the excitation energy after the maximum or peak of the excitation pulse, which can allow for shorter lifetime detection. good. Such techniques may electrically drive the excitation source to reduce excitation power after peak power. The electrical drive signal may be adjusted to drive the intensity of the pulse of excitation energy to zero as quickly as possible after the peak pulse. Such techniques may include reversing the sign of the electrical drive signal after peak power is generated. The electrical signal can be tuned to rapidly reduce carrier density after the first relaxation oscillation or first oscillation of the optical signal. By reducing the carrier density after the first oscillation, a light pulse of only the first oscillation may be generated. The electrical signal may be configured to produce a short pulse that quickly turns off the optical signal by reducing the number of photons emitted after the peak of the signal. Picosecond laser diode systems may be designed to emit pulses of light, according to some embodiments. In some embodiments, saturable absorbers, including semiconductor saturable absorbers (SESAM), may be used to suppress optical tails. In such embodiments, the use of a saturable absorber may suppress the optical tail by 3-5 dB, or even more than 5 dB in some cases. Reducing the effects of the tail in the excitation pulse reduces and/or eliminates any requirement for additional filtering of the excitation energy, increases the range of measurable lifetimes, and/or allows for faster pulse rates. can make it possible. Increasing the excitation pulse rate allows more experiments to be performed in a given time period, thereby obtaining sufficient statistics to identify the lifetime of the markers labeling the sample. The time required can be shortened.

Additionally, two or more of these techniques may be used together to generate pulsed excitation energy. For example, pulsed excitation energy emitted from a directly modulated source may be further modified using optical modulation techniques. Techniques for modulating the excitation pulse and adjusting the electrical pulse drive signal may be combined in any suitable manner to optimize the pulse excitation energy for performing lifetime measurements. A modulated electrical drive signal may be added to the pulsed excitation energy from a directly modulated source.

In some embodiments, a laser diode with a certain number of wire bonds may be used as a pulsed excitation source. A laser diode with more wire bonds can reduce the inductance of the pump source. A laser diode with a lower inductance will allow the current to the laser to operate at a higher frequency. Choosing a packaging method to minimize inductance improves the power delivered to the excitation source at higher frequencies, allowing for shorter excitation pulses, more rapid decay of optical power after the peak and /or allow for increased pulse repetition rates for detecting luminescent markers.

In some embodiments, a transmission circuit in combination with an excitation source may be used to generate light pulses. The transmission circuit may match the impedance of the laser diode to improve the performance and/or quality of the optical pulses. In some embodiments, the transmission circuit impedance may be 50 ohms. In some examples, the termination resistance may be similar to the resistance of the line to avoid reflections. Alternatively or additionally, the termination impedance may be similar to the impedance of the line to avoid reflections. The termination impedance may be less than the line impedance to reflect negative pulses. In other embodiments, the terminating impedance may have capacitive or inductive components to control the shape of the negative reflected pulse. In other embodiments, the transmission circuit may allow higher frequency pulses. A transmission circuit may be used to generate electrical pulses having a frequency in the range of 40 MHz to 500 MHz. A transmission circuit may be used in combination with the regulated electrical signals described above to generate a pulsed light source having light pulses with certain time intervals and specific time intervals.

Techniques for conditioning the electrical signal to improve the generation of light pulses may involve connecting the excitation source to a circuit with negative bias capability. In some embodiments, a negative bias may be provided to the excitation source after the light pulse is emitted to reduce tail emission in the light pulse. Exemplary circuits may include current sources, diode lasers, resistors, capacitors, and switches that may be implemented to reduce the presence of tails in light pulses. Such a circuit may generate a constant current that bypasses the diode laser when the switch is closed or conducting. When the switch is open, the switch may have a high resistance and current may flow through the diode laser. Light pulses may be generated by opening and closing a switch to supply an intermittent current to the diode laser. In some examples, the resistor is high enough and the capacitor is small enough so that there is a voltage across the capacitor when the switch opens and the diode laser fires. When the switch is closed, the voltage across the capacitor reverse biases the diode laser. Such reverse biasing may reduce or eliminate the presence of tails in the light pulse. In such a case, the switch may be configured to close after the peak of the light pulse to decrease the laser power just after the peak light pulse. The values of the resistors in the circuit can be chosen such that the charge on the capacitor discharges before the switch is subsequently opened and/or a subsequent light pulse is generated by the laser diode.

Additional circuitry may be provided to condition the electrical signal of the laser diode to generate the light pulses. In some embodiments, multiple capacitors, resistors, and voltages may be connected in a network circuit to control the waveform of the electrical signal supplied to the laser diode. The controlled waveforms are the corresponding signals S1, S2, . . . , SN determine a number of voltages V1, V2, . . . , VN.

In some embodiments, electrical signals for generating light pulses may use circuits with discrete components, including radio frequency (RF) and/or microwave components. Discrete components that may be included in such circuits are DC blocks, adapters, logic gates, terminators, phase shifters, delays, attenuators, combiners, and/or RF amplifiers. Such components may be used to generate a positive electrical signal with one constant amplitude followed by a negative electrical signal with another amplitude. There may be a delay between positive and negative electrical signals. In other embodiments, the circuit may generate multiple electrical signals that are combined to form an electrical pulse signal configured to drive the excitation source. Such circuits can produce a differential output that can be used to increase the power of optical pulses. By adjusting individual components of the circuit, the electrical output signal may be adjusted to produce light pulses suitable for lifetime measurements.

In some embodiments, excitation sources may be combined to generate light pulses for lifetime measurements. A synchronized pulse source may be coupled to a circuit or load over a certain distance. In some embodiments, the excitation sources may be coupled in parallel to the circuit. The excitation sources may be from the same source or from multiple sources. In some embodiments with multiple sources, the multiple sources may differ in the type of excitation source. When combining sources, it may be important to consider the impedance of the circuit and the excitation source in order to provide sufficient power to the excitation source. Combining sources may be accomplished by using one or more of the techniques described above for generating pulsed excitation sources.

The excitation source may comprise a battery or any other power source arranged to power the excitation source. For example, the excitation source may be located within the base instrument and its operating power may be received through the integrated bioanalytical device to which it is coupled (eg, via conductive power leads). The excitation source may be controlled independently or in conjunction with control of the integrated bioanalytical device. By way of example only, excitation source control signals may be provided to the excitation source wirelessly with a personal computer and/or integrated bioanalytical device or via a wired interconnect (eg, a USB interconnect).

In some implementations, the excitation source may operate in time gated and/or synchronized with one or more sensors of the integrated device. For example, the excitation source may be turned on to excite the luminescent marker and then turned off. The sensor may be turned off while the excitation source is turned on, and then turned on during the sampling interval after the excitation source is turned off. In some embodiments, the sensor may be turned on for sampling intervals while the excitation source is turned on.

IV. Examples of Measurements with Integrated Devices and Excitation Sources Measurements for detecting, analyzing and/or probing molecules in a sample use integrated devices or any combination of integrated devices and excitation sources described in this application. can be obtained by The excitation source may be a pulsed excitation source or, in some cases, a continuous wave source. A luminescent marker tagged to a particular sample can indicate the presence of the sample. Luminescent markers can be identified by excitation energy, emission emission wavelength, and/or lifetime of the radiant energy emitted by the marker. Markers with similar emission emission wavelengths can be identified by determining the lifetime of each marker. Additionally, markers with similar lifetimes may be identified by the emission emission wavelength for each marker. Quantitative analysis and/or identification of markers and associated samples may be performed by using markers that are identified by a combination of temporal and/or spectral properties of emitted luminescence.

A lifetime measurement may be used to determine the presence of a marker in a sample well. The lifetime of a luminescent marker can be identified by performing multiple experiments in which the luminescent marker is excited to an excited state and then the time for photon emission is measured. The excitation source is pulsed to produce a pulse of excitation energy and directed at the marker. The time between the excitation pulse and the subsequent photon emission event from the luminescent marker is measured. Such experiments may be repeated with multiple excitation pulses to determine the number of instances in which a photon is emitted within a particular time interval. Such results can populate a histogram representing the number of photon emission events occurring within a series of discrete time intervals or time bins. The number of time bins and/or the time interval between each bin can be adjusted to identify a particular set of lifespans and/or markers.

Following is a description of example measurements that, in some embodiments, may be performed to identify luminescent markers. Specifically, consider examples of identifying luminescence markers using only luminescence lifetime measurements, binding spectra and luminescence lifetime measurements, and luminescence lifetime measurements only, but using two different excitation energies. Embodiments are not limited to the examples detailed below. For example, some embodiments may identify luminescent markers using spectral measurements alone.

Any suitable luminescent marker may be used. In some embodiments, commercially available fluorophores may be used. By way of example and not limitation, the following fluorophores may be used: Atto Rho14 (“ATRho14”), DyLight® 650 (“D650”), Seta™ Tau 647 (“ ST647”), CF™ 633 (“C633”), CF™ 647 (“C647”), Alexa Fluor® 647 (“AF647”), BODIPY® 630/650 (“B630 ”), CF™ 640R (“C640R”) and/or Atto 647N (“AT647N”).

Additionally and/or optionally, the luminescent markers may be modified in any suitable manner to enhance the speed and accuracy of the sample analysis process. For example, a photostabilizer may be conjugated to a luminescent marker. Examples of light stabilizers include, but are not limited to, oxygen scavengers or triplet quenchers. Conjugation of a photostabilizer to a luminescent marker may increase the rate of emitted photons and may reduce the "blinking" effect in which the luminescent marker does not emit photons. In some embodiments, the increased rate of photon emission may increase the probability of detection of the biological event if the biological event occurs on the millisecond scale. An increase in the rate of photon events, in turn, increases the signal-to-noise ratio of the luminescence signal, increasing the speed at which lifetime measurements are made, resulting in faster and more accurate sample analysis.

Additionally, the environment within the sample well of the integrated device may be adjusted to engineer the lifetime of the markers, if desired. This can be achieved by recognizing that marker longevity is driven by the marker's density of states, which can be adjusted using the environment. For example, the farther the marker is from the bottom metal layer of the sample well, the longer the lifetime. Therefore, the depth of the bottom of the sample well, such as the divot, may extend a certain distance from the metal layer in order to increase the lifetime of the marker. Also, the materials used to form the sample wells can affect the longevity of the markers. Different markers are typically shifted in their lifespans in the same direction (eg, longer or shorter), but can affect different markers to different degrees. Thus, two markers that are indistinguishable via lifetime measurements in free space may be designed to be distinguishable by creating a sample well environment to modulate the lifetime of the various markers.

A. Lifetime Measurements Lifetime measurements may be performed using one excitation energy wavelength to excite the markers in the sample well. To discriminate between individual markers based on lifespan measurements, a combination of markers with distinct lifespans is selected. Furthermore, the combination of markers can reach an excited state when illuminated by the excitation source used. An integrated device configured for lifetime measurements using one excitation may comprise a plurality of pixels arranged along rows with each sample well configured to couple to the same waveguide. good. A pixel comprises a sample well and a sensor. One or more microcavities or bull's eye gratings may be used to couple the waveguides to the sample well of each pixel.

The pulsed excitation source may be one of the pulsed excitation sources using the techniques described above. In some cases, the pulsed excitation source may be a semiconductor laser diode configured to emit pulses through direct modulated electrical pumping of the laser diode. The power of the pulse is less than 20 dB of the pulse peak power at approximately 250 picoseconds after the peak. The time interval between each excitation pulse is in the range of 20-200 picoseconds. The duration between each excitation pulse is in the range of 1-50 ns. A schematic diagram of how an example measurement may be performed is presented in FIG. 31A. Since one excitation energy is used, excitation filters suitable for reducing the transmission of excitation energy to the sensor may be formed into an integrated device, such as the wavelength excitation filters discussed above.

Each pixel sensor has at least one photosensitive area per pixel. A photon is detected within the time interval in which the photon reaches the sensor. Increasing the number of time bins can improve the resolution of the recorded histogram of photons collected over a series of time bins and improve the differences between different luminescent markers. If the sensor is configured to detect specific wavelengths, the four luminescent markers may emit emissions similar to the specific wavelengths. Alternatively, the four luminescent markers can emit at different wavelengths.

An example of a set of four luminescent markers distinguishable based on lifetime measurements are ATRho14, Cy®5, AT647N, and CF™633, as represented by the plot in FIG. 31B. These four markers have different lifetimes and produce distinguishable histograms when at least four time bins are used. Figure 31C outlines the signal profile for each of these markers over 16 time bins. Signal profiles are normalized for each marker. The time bins vary in time interval to provide a unique signal profile for each of the markers. Figures 32A and 32B show both continuous and discrete signal profiles of another exemplary set of distinguishable markers, ATTO Rho14, D650, ST647 and CF™633, respectively, based on lifespan measurements. is illustrated. Other sets of markers include ATTO Rho14, C647, ST647, CF™ 633; Alexa Fluor® 647, B630, C640R, CF™ 633; and ATTO Rho14, ATTO 647N, AlexaFluor647, CF ( trademark) 633.

B. Spectral-Lifetime Measurements Lifetime measurements may be combined with spectral measurements of one or more luminescent markers. Spectral measurements depend on the wavelength of emission of individual markers and are captured using at least two sensor areas per pixel. An exemplary construction of an integrated device comprises a pixel each having a sensor with two distinct regions, each configured to detect different wavelengths. Multi-wavelength filters may be used to selectively transmit different wavelengths of light to each sensor area. For example, one sensor area and filter combination may be configured to detect red light, while another sensor area and filter combination may be configured to detect green light.

Combining both lifetime and spectral measurements may be performed using one excitation energy wavelength to excite the markers in the sample well. A combination of markers with at least two distinct emission wavelengths is selected, markers that emit at a wavelength with distinct lifetimes are selected, and discrimination is made between individual markers based on lifetime and spectral measurements. Furthermore, combinations of markers are chosen such that they can reach an excited state when illuminated by the excitation source used.

The excitation source is a pulsed excitation source and may be one of the excitation sources using the techniques described above. In some cases, the pulsed excitation source may be a semiconductor laser diode configured to emit pulses through direct modulated electrical pumping of the laser diode. The power of the pulse is less than 20 dB peak power 250 picoseconds after the peak. The duration of each excitation pulse is in the range of 20-200 picoseconds. The time interval between each excitation pulse is in the range of 1-50 ns. A schematic diagram of how an example measurement may be performed is shown in FIG. 33A. Since one excitation energy is used, an excitation filter suitable to reduce the transmission of excitation energy to the sensor may be used.

Each pixel sensor has at least two photosensitive areas per pixel. In some embodiments, there are two photosensitive areas per pixel. In other embodiments, there are four photosensitive areas per pixel. Each photosensitive area is configured to detect a different wavelength or range of wavelengths. Photons are detected within the time interval of reaching the sensor. Increasing the number of time bins may improve the resolution of the recorded histogram of photons collected over a series of time bins and improve the differences between luminescence markers that differ by their individual lifetimes. In some embodiments, there are two time bins per area of the sensor. In other embodiments, there are four time bins per region of the sensor.

An example of a set of four luminescent markers distinguishable based on lifetime measurements is ATTO Rho14, AS635, AlexaFluor® 647, and ATTO 647N. These four markers have one similar wavelength and two markers emitting at another similar wavelength. Within each pair of markers emitting at similar wavelengths, the pair of markers have different lifetimes when at least four time bins are used, producing a distinguishable histogram. In this example, ATTO Rho14 and AS635 emit similar emission wavelengths and have distinct lifetimes. AlexaFluor® 647 and ATTO 647N emit similar emission wavelengths, different from those emitted by ATTO Rho 14 and AS635, and have distinct lifetimes. FIG. 33B presents a plot of lifetime as a function of emission wavelength for this set of markers, illustrating how each of these markers is identifiable based on the combination of lifetime and emission wavelength. FIG. 34A presents plots of power as a function of wavelength for ATT Rho 14, Alexa Fluor® 647, and ATT) 647N. FIG. 34B presents a plot of fluorescence signal over time for each one of these markers when present in a sample well with a diameter of 135 nm. Figure 35A illustrates the signal profile of these markers across four sensitive regions, each region capturing four time bins. The signal profile is normalized and used to distinguish between different markers by the relative number of photons captured by the photosensitive region for each of the four time bins. Another set of four fluorophores for such spectral lifetime measurements are ATRho14, D650, ST647, CF™633; ATTO Rho14, C647, ST647, CF™633; 647, B630, C640R, CF™633; and ATTO Rho 14, ATTO 647N, Alexa Fluor® 647, CF™633. FIG. 35B presents a plot of the intensity signal profile over time for ATRho14, D650, ST647, and C633. FIG. 35C illustrates the signal profile of ATRho14.

C. Lifetime-Excitation Energy Measurements Lifetime measurements in combination with using at least two excitation energy wavelengths may be used to discriminate between multiple markers. If one excitation wavelength is used and another excitation wavelength is not used, several markers can be excited. To distinguish between individual markers based on lifetime measurements, a combination of markers with distinct lifetimes is selected for each excitation wavelength. In this embodiment, the integrated device may be configured with a sensor having one area for each pixel, and the external excitation source is an electrically modulated pulsed diode laser with temporal interleaving. It may be configured to provide certain two excitation energy wavelengths.

An excitation source is a combination of at least two excitation energies. The excitation source is a pulsed excitation source and may be one or more excitation sources using the techniques described above. In some examples, the pulsed excitation source may be two semiconductor laser diodes configured to emit pulses through direct modulated electrical pumping of the laser diodes. The power of the pulse is 20 dB below the pulse peak power at 250 picoseconds after the peak. The time interval between each excitation pulse is in the range of 20-200 picoseconds. The time interval between each excitation pulse is in the range of 1-50 ns. One excitation wavelength is emitted for one pulse, and knowledge of the excitation wavelength uniquely identifies a subset of markers with well-defined lifetimes. In some embodiments, pulses of excitation alternate between different wavelengths. For example, if two excitation wavelengths are used, subsequent pulses alternate between one wavelength and the other. A schematic diagram of how an example measurement may be performed is presented in FIG. 36A. Any suitable technique for combining multiple excitation sources with different wavelengths and interleaving pulses may be used. Several examples of techniques for delivering pulses of one or more excitation wavelengths to the illustrated array of sample wells are described herein. In some embodiments, there is a single waveguide per row of sample wells and two excitation sources combined so that the pulses of excitation energy alternate between the two excitation wavelengths. In some embodiments, there are two waveguides per row of sample wells, each waveguide configured to carry one of the two excitation wavelengths. In other embodiments, there is a single waveguide per row of pixels, with one wavelength coupled to one end of the waveguide and another wavelength coupled to the other end.

Each pixel sensor has at least one photosensitive area per pixel. The photosensitive area may have dimensions of 5 microns by 5 microns. Photons are detected within the time interval of reaching the sensor. Increasing the number of time bins can improve the resolution of the recorded histogram of photons collected over a series of time bins and improve the differences between different luminescent markers. The sensor has at least two time bins.

An example of a set of four luminescent markers identifiable based on lifetime measurements is AlexaFluor® 546, Cy® 3B, AlexaFluor® 647, and ATTO 647N. As depicted in FIG. 36B, Alexa Fluor® 546 and Cy® 3B excite at one wavelength, eg, 532 nm, and have well-defined lifetimes. Alexa Fluor® 647 and ATTO 647N excite at another wavelength of 640 nm and have distinct lifetimes as depicted in FIG. 37A. Distinguishable normalized signal profiles over 16 time bins for ATTO647N and CF™333, both excited at 640 nm, are presented in FIG. 37B. By detecting photons after a known excitation wavelength, one of these two paired markers is determined based on the previous excitation wavelength and each marker in the pair is identified based on lifetime measurements.

Equivalents and Scope Various aspects of the present application may be used singly, in combination, or in various arrangements not specifically discussed in the foregoing embodiments, and therefore, in their application, should not be construed as having the foregoing details. It is not limited in its application to the details and arrangements of components shown in the description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

Also, the present invention may be embodied as a method, of which at least one embodiment is provided. Acts performed as part of a method may be ordered in any suitable manner. Thus, although depicted as sequential acts in the illustrated embodiment, embodiments may be constructed in which the acts are performed in a sequence not illustrated, which may include performing several acts simultaneously. good.

The use of ordinal terms such as “first,” “second,” “third,” etc. in a claim to modify claim elements does, by itself, act in a certain way. When using the same claim elements to identify the claim elements, rather than implying that any sequential priority, weight priority, or order of one claim element precedes another or chronological order. It is used only as a marker to distinguish one claim element having a given name from another element having a name (with the exception of the use of ordinal terminology).

Also, the terminology and terminology used herein are for the purpose of description and should not be regarded as limiting. "including", "comprising" or "having", "containing", "including" )”, and variations thereof, are meant to encompass the items listed thereafter and their equivalents and additional items.

Claims (21)

A method for determining the sequence of a template nucleic acid , comprising:
(i) exposing the complexes in the target volume to multiple types of luminescently labeled nucleotides, said complexes comprising template nucleic acids, primers, and polymerizing enzymes, each type of luminescently labeled nucleotides comprising: having at least one of different luminescence lifetimes and luminescence intensities;
(ii) directing a series of one or more pulses of excitation energy into the vicinity of said target volume;
(iii) detecting multiple emitted photons from luminescently labeled nucleotides during sequential incorporation into a nucleic acid comprising said primer;
(iv) for each detected emitted photon, determining the elapsed time between a prior pulse of excitation energy and detection of said emitted photon;
(v) identifying the sequence of the incorporated nucleotide by determining the emission lifetime and emission intensity of said luminescently labeled nucleotide, wherein said emission lifetime is equal to said prior excitation energy for a plurality of emitted photons; determined based on the elapsed time between detection of emitted photons, wherein the luminescence intensity is determined based on the number of emitted photons per unit time.
How to prepare. 2. The method of claim 1, wherein each type of luminescently labeled nucleotide emits photons after being irradiated with a single excitation energy. 3. The method of claim 2, wherein the single excitation energy is in the spectral range of about 470 to about 510 nm, about 510 to about 550 nm, about 550 to about 580 nm, about 580 nm to about 620 nm, or about 620 nm to about 670 nm. Method. After irradiation with a first excitation energy, one or more types of luminescently labeled nucleotides emit photons, and after irradiation with a second excitation energy, one or more types of luminescently labeled nucleotides emit photons. 2. The method of claim 1, wherein the radiating wherein said first excitation energy is in the spectral range of about 470 nm to about 510 nm, about 510 nm to about 550 nm, about 550 to about 580 nm, about 580 nm to about 620 nm, or about 620 nm to about 670 nm; is in the spectral range of about 470 to about 510 nm, about 510 to about 550 nm, about 550 to about 580 nm, about 580 nm to about 620 nm, or about 620 nm to about 670 nm. A method according to any one of claims 1 to 5, wherein each type of luminescently labeled nucleotide emits photons in a single spectral range. Claims 1-, wherein the one or more types of luminescently labeled nucleotides emit photons in a first spectral range and the one or more types of luminescently labeled nucleotides emit photons in a second spectral range. 6. The method of any one of 5. at least one type, at least two types, at least three types, or at least four of said types of luminescently labeled nucleotides are each 6-TAMRA, 5/6-Carboxyrhodamine 6G, Alex Fluor 546, Alexa Fluor ) 555, Alexa Fluor® 568, Alexa Fluor® 610, Alexa Fluor® 647, Aberrior Star 635, ATTO425, ATTO465, ATTO488, ATTO495, ATTO514, ATTO520, ATTO Rho6G, ATTO542, ATTO647N, ATTO Rho 14, Chromis 630, Chromis 654A, Chromeo™ 642, CF™ 514, CF™ 532, CF™ 543, CF™ 546, CF™ 546, CF™ 555, CF ( 568, CF 633, CF 640R, CF 660C, CF 660R, CF 680R, Cy 3, Cy 3B, Cy ( Registered Trademark) 3.5, Cy (Registered Trademark) 5, Cy (Registered Trademark) 5.5, Dyomics-530, Dyomics-547P1, Dyomics-549P1, Dyomics-550, Dyomics-554, Dyomics-555, Dyomics-556 , Dyomics-560, Dyomics-650, Dyomics-680, DyLight® 554-R1, DyLight® 530-R2, DyLight® 594, DyLight® 635-B2, DyLight® Trademark) 650, DyLight® 655-B4, DyLight® 675-B2, DyLight® 675-B4, DyLight® 680, HiLyte® Fluor 532, HiLyte® Fluor 555, is selected from the group consisting of HiLyte™ Fluor 594, LightCycler® 640R, Seta™ 555, Seta™ 670, Seta™ 700, Seta™ u647, and Seta™ u665 comprising a luminescent label or of the formula (dye 101), (dye 102), (dye 103), (dye 104), (dye 105), or (dye 106);


A method according to any one of claims 1 to 7, wherein each sulfonate or carboxylate is independently optionally protonated. The method of any one of claims 1-8, wherein the series of pulses is applied at an average frequency of about 10 MHz to about 100 MHz or about 100 MHz to about 1 GHz. 10. The method of claim 9, wherein said average frequency is between about 50 MHz and about 200 MHz. 11. The method of any one of claims 1-10, wherein the number of detected emitted photons is from about 10 to about 100 or from about 100 to about 1,000 for each incorporated luminescent-labeled nucleotide. the method of. The method of any one of claims 1-11, wherein the complex comprises a DNA polymerase. The method of any one of claims 1-12, wherein the complex is immobilized on a solid support. 14. The method of claim 13, wherein said solid support is the bottom surface of a sample well. A method according to any one of claims 1 to 14, wherein a single complex is localized to said target volume. wherein said plurality of types of luminescently labeled nucleotides comprises a first nucleotide comprising deoxyadenosine, a second nucleotide comprising deoxyguanosine, a third nucleotide comprising deoxycytidine, and a fourth nucleotide comprising deoxythymidine. Item 16. The method according to any one of items 1 to 15. 17. The method of any one of claims 1-16, wherein each of said plurality of types of luminescent labeled nucleotides comprises a luminescent label and a nucleotide connected by a linker. 18. The method of claim 17, wherein said linker comprises a protective molecule that minimizes the extent of interaction between said luminescent label and said polymerizing enzyme. 19. The method of claim 18, wherein said protective molecule comprises a protein or nucleic acid. 20. The method of any one of claims 1-19, wherein at least one type of luminescently labeled nucleotide comprises first and second chromophores of a FRET pair. 21. The method of claim 20, wherein the FRET pair absorbs excitation energy in the same spectral range as one or more of the plurality of other luminescent labeled molecules.

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